Methods for diagnosing and treating heart disease

ABSTRACT

The invention provides methods of diagnosing heart disease, such as heart failure, methods for identifying compounds that can be used to treat or to prevent heart disease, and methods of using these compounds to treat or to prevent heart disease. Also provided in the invention are animal model systems that can be used in screening methods.

FIELD OF THE INVENTION

[0001] This invention relates to methods for diagnosing and treatingheart disease.

BACKGROUND OF THE INVENTION

[0002] Heart disease is a general term used to describe many differentheart conditions. For example, coronary artery disease, which is themost common heart disease, is characterized by constriction or narrowingof the arteries supplying the heart with oxygen-rich blood, and can leadto myocardial infarction, which is the death of a portion of the heartmuscle. Heart failure is a condition resulting from the inability of theheart to pump an adequate amount of blood through the body. Heartfailure is not a sudden, abrupt stop of heart activity but, rather,typically develops slowly over many years, as the heart gradually losesits ability to pump blood efficiently. Risk factors for heart failureinclude coronary artery disease, hypertension, valvular heart disease,cardiomyopathy, disease of the heart muscle, obesity, diabetes, and afamily history of heart failure.

SUMMARY OF THE INVENTION

[0003] The invention provides diagnostic, drug screening, andtherapeutic methods that are based on the observation that a mutation ina zebrafish gene, designated heart of glass (heg), leads to a phenotypein zebrafish that is similar to heart failure in mammals.

[0004] In a first aspect, the invention provides a method of determiningwhether a test subject (e.g., a mammal, such as a human) has, or is atrisk of developing, a disease or condition related to a heart of glassprotein (e.g., heart disease, such as heart failure). This methodinvolves analyzing a nucleic acid molecule of a sample from the testsubject to determine whether the test subject has a mutation (e.g., theheart of glass mutation) in a gene encoding the protein. The presence ofa mutation indicates that the test subject has, or is at risk ofdeveloping, a disease related to a heart of glass protein. This methodcan also involve the step of using nucleic acid molecule primersspecific for a gene encoding a heart of glass protein for nucleic acidmolecule amplification of the gene by the polymerase chain reaction. Itcan also involve sequencing a nucleic acid molecule encoding a heart ofglass protein from said test subject.

[0005] In a second aspect, the invention provides a method foridentifying a compound that can be used to treat or to prevent heartdisease (e.g., heart failure). This method involves contacting anorganism (e.g., a zebrafish) having a mutation (e.g., the heart of glassmutation) in a gene encoding a heart of glass protein and having aphenotype characteristic of heart disease with the compound, anddetermining the effect of the compound on the phenotype. Detection of animprovement in the phenotype indicates the identification of a compoundthat can be used to treat or to prevent heart disease.

[0006] In a third aspect, the invention provides a method of treating orpreventing heart disease (e.g., heart failure) in a patient (e.g., apatient having a mutation (e.g., the heart of glass mutation) in a geneencoding a heart of glass protein), involving administering to thepatient a compound identified using the method described above.

[0007] In a fourth aspect, the invention provides of treating orpreventing heart disease in a patient. This method involvesadministering to the patient a functional heart of glass protein or anexpression vector including a nucleic acid molecule encoding theprotein.

[0008] In a fifth aspect, the invention includes a substantially purezebrafish heart of glass polypeptide. This polypeptide can include orconsist essentially of, for example, an amino acid sequence that issubstantially identical to the amino acid sequence of SEQ ID NO:2 or SEQID NO:3.

[0009] In a sixth aspect, the invention provides a substantially purenucleic acid molecule (e.g., a DNA molecule) including a sequenceencoding a zebrafish heart of glass polypeptide. This nucleic acidmolecule can encode a polypeptide including or consisting essentially ofan amino sequence that is substantially identical to the amino acidsequence of SEQ ID NO:2 or SEQ ID NO:3.

[0010] In a seventh aspect, the invention provides a vector includingthe nucleic acid molecule described above.

[0011] In an eighth aspect, the invention includes a cell including thevector described above.

[0012] In a ninth aspect, the invention provides a non-human transgenicanimal (e.g., a zebrafish) including the nucleic acid molecule describedabove.

[0013] In a tenth aspect, the invention provides a non-human animalhaving a knockout mutation in one or both alleles encoding a heart ofglass polypeptide.

[0014] In an eleventh aspect, the invention includes a cell from thenon-human knockout animal described above.

[0015] In a twelveth aspect, the invention includes a non-humantransgenic animal (e.g., a zebrafish) including a nucleic acid moleculeencoding a mutant heart of glass polypeptide, e.g., a polypeptide havingthe heart of glass mutation.

[0016] In a thirteenth aspect, the invention provides an antibody thatspecifically binds to a heart of glass polypeptide.

[0017] In a fourteenth aspect, the invention provides the use of acompound identified using the method described above in the preparationof a medicament for treating or preventing heart disease in a patient.

[0018] In a fifteenth aspect, the invention provides the use of a heartof glass protein or an expression vector including a nucleic acidmolecule encoding this protein in the preparation of a medicament fortreating or preventing heart disease in a patient.

[0019] By “polypeptide” or “polypeptide fragment” is meant a chain oftwo or more amino acids, regardless of any post-translationalmodification (e.g., glycosylation or phosphorylation), constituting allor part of a naturally or non-naturally occurring polypeptide. By“post-translational modification” is meant any change to a polypeptideor polypeptide fragment during or after synthesis. Post-translationalmodifications can be produced naturally (such as during synthesis withina cell) or generated artificially (such as by recombinant or chemicalmeans). A “protein” can be made up of one or more polypeptides.

[0020] By “heart of glass protein” or “heart of glass polypeptide” ismeant a polypeptide that has at least 45%, preferably at least 60%, morepreferably at least 75%, and most preferably at least 90% amino acidsequence identity to the sequence of a human (see, e.g., SEQ ID NO:5) ora zebrafish (see, e.g., SEQ ID NO:2 or SEQ ID NO:3) heart of glasspolypeptide. Polypeptide products from splice variants (e.g., heg1 andheg2) of heart of glass gene sequences and heart of glass genescontaining mutations are also included in this definition. A heart ofglass polypeptide as defined herein plays a role in heart development,modeling, and function. It can be used as a marker of heart disease,such as heart failure. The invention thus includes proteins having anyof these and other functions of heart of glass, as described herein, andhaving sequence identity (e.g., at least 75%, 85%, 90%, or 95%) to ahuman or a zebrafish (SEQ ID NO:2 or SEQ ID NO:3) heart of glasspolypeptide.

[0021] By a “heart of glass nucleic acid molecule” is meant a nucleicacid molecule, such as a genomic DNA, cDNA, or RNA (e.g., mRNA)molecule, that encodes a heart of glass protein (e.g., a human (encodedby, e.g., SEQ ID NO:4) or a zebrafish (encoded by, e.g., SEQ ID NO:1)heart of glass protein), a heart of glass polypeptide, or a portionthereof, as defined above. A mutation in a heart of glass nucleic acidmolecule can be characterized, for example, by a G to A nucleotidetransversion at position 497, predicting a change from a tryptophancodon to a stop codon (TGG->TAG). In addition to these zebrafish heartof glass mutations, the invention includes any mutation that results inaberrant heart of glass protein production or function, including, onlyas examples, null mutations and mutations causing truncations.

[0022] The term “identity” is used herein to describe the relationshipof the sequence of a particular nucleic acid molecule or polypeptide tothe sequence of a reference molecule of the same type. For example, if apolypeptide or a nucleic acid molecule has the same amino acid ornucleotide residue at a given position, compared to a reference moleculeto which it is aligned, there is said to be “identity” at that position.The level of sequence identity of a nucleic acid molecule or apolypeptide to a reference molecule is typically measured using sequenceanalysis software with the default parameters specified therein, such asthe introduction of gaps to achieve an optimal alignment (e.g., SequenceAnalysis Software Package of the Genetics Computer Group, University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705, BLAST, or PILEUP/PRETTYBOX programs). These software programsmatch identical or similar sequences by assigning degrees of identity tovarious substitutions, deletions, or other modifications. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine, valine, isoleucine, and leucine; asparticacid, glutamic acid, asparagine, and glutamine; serine and threonine;lysine and arginine; and phenylalanine and tyrosine.

[0023] A nucleic acid molecule or polypeptide is said to be“substantially identical” to a reference molecule if it exhibits, overits entire length, at least 51%, preferably at least 55%, 60%, or 65%,and most preferably 75%, 85%, 90%, or 95% identity to the sequence ofthe reference molecule. For polypeptides, the length of comparisonsequences is at least 16 amino acids, preferably at least 20 aminoacids, more preferably at least 25 amino acids, and most preferably atleast 35 amino acids. For nucleic acid molecules, the length ofcomparison sequences is at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably at least 110 nucleotides.

[0024] A heart of glass nucleic acid molecule or a heart of glasspolypeptide is “analyzed” or subject to “analysis” if a test procedureis carried out on it that allows the determination of its biologicalactivity or whether it is wild type or mutated. For example, one cananalyze the heart of glass genes of an animal (e.g., a human or azebrafish) by amplifying genomic DNA of the animal using the polymerasechain reaction, and then determining whether the amplified DNA containsa mutation, for example, the heart of glass mutation, by, e.g.,nucleotide sequence or restriction fragment analysis.

[0025] By “probe” or “primer” is meant a single-stranded DNA or RNAmolecule of defined sequence that can base pair to a second DNA or RNAmolecule that contains a complementary sequence (“target”). Thestability of the resulting hybrid depends upon the extent of the basepairing that occurs. This stability is affected by parameters such asthe degree of complementarity between the probe and target molecule, andthe degree of stringency of the hybridization conditions. The degree ofhybridization stringency is affected by parameters such as thetemperature, salt concentration, and concentration of organic molecules,such as formamide, and is determined by methods that are well known tothose skilled in the art. Probes or primers specific for heart of glassnucleic acid molecules, preferably, have greater than 45% sequenceidentity, more preferably at least 55-75% sequence identity, still morepreferably at least 75-85% sequence identity, yet more preferably atleast 85-99% sequence identity, and most preferably 100% sequenceidentity to the sequences of human or zebrafish (SEQ ID NO:1) heart ofglass genes (either strand). Preferably, the probes or primers bind toheart of glass genes (or the complements thereof) under highly stringentconditions, as described herein.

[0026] Probes can be detectably labeled, either radioactively ornon-radioactively, by methods that are well known to those skilled inthe art. Probes can be used for methods involving nucleic acidhybridization, such as nucleic acid sequencing, nucleic acidamplification by the polymerase chain reaction, single strandedconformational polymorphism (SSCP) analysis, restriction fragmentpolymorphism (RFLP) analysis, Southern hybridization, northernhybridization, in situ hybridization, electrophoretic mobility shiftassay (EMSA), and other methods that are well known to those skilled inthe art.

[0027] A molecule, e.g., an oligonucleotide probe or primer, a gene orfragment thereof, a cDNA molecule, a polypeptide, or an antibody, can besaid to be “detectably-labeled” if it is marked in such a way that itspresence can be directly identified in a sample. Methods for detectablylabeling molecules are well known in the art and include, withoutlimitation, radioactive labeling (e.g., with an isotope, such as ³²P or³⁵S) and nonradioactive labeling (e.g., with a fluorescent label, suchas fluorescein).

[0028] By a “substantially pure polypeptide” is meant a polypeptide (ora fragment thereof) that has been separated from proteins and organicmolecules that naturally accompany it. Typically, a polypeptide issubstantially pure when it is at least 60%, by weight, free from theproteins and naturally occurring organic molecules with which it isnaturally associated. Preferably, the polypeptide is a heart of glasspolypeptide that is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight, pure. A substantially pure heart ofglass polypeptide can be obtained, for example, by extraction from anatural source (e.g., isolated heart tissue), by expression of arecombinant nucleic acid molecule encoding a heart of glass polypeptide,or by chemical synthesis. Purity can be measured by any appropriatemethod, e.g., by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

[0029] A polypeptide is substantially free of naturally associatedcomponents when it is separated from those proteins and organicmolecules that accompany it in its natural state. Thus, a protein thatis chemically synthesized or produced in a cellular system that isdifferent from the cell in which it is naturally produced issubstantially free from its naturally associated components.Accordingly, substantially pure polypeptides not only include those thatare derived from eukaryotic organisms, but also those synthesized in E.coli or other prokaryotes.

[0030] An antibody is said to “specifically bind” to a polypeptide if itrecognizes and binds to the polypeptide (e.g., a heart of glasspolypeptide), but does not substantially recognize and bind to othermolecules (e.g., non-heart of glass related polypeptides) in a sample,e.g., a biological sample that naturally includes the polypeptide.

[0031] By “high stringency conditions” is meant conditions that allowhybridization comparable with the hybridization that occurs using a DNAprobe of at least 500 nucleotides in length, in a buffer containing 0.5M NaHPO₄, pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA (fraction V), at atemperature of 65° C., or a buffer containing 48% formamide, 4.8×SSC,0.2 M Tris-Cl, pH 7.6, 1× Denhardt's solution, 10% dextran sulfate, and0.1% SDS, at a temperature of 42° C. (These are typical conditions forhigh stringency northern or Southern hybridizations.) High stringencyhybridization is also relied upon for the success of numerous techniquesroutinely performed by molecular biologists, such as high stringencyPCR, DNA sequencing, single strand conformational polymorphism analysis,and in situ hybridization. In contrast to northern and Southernhybridizations, these techniques are usually performed with relativelyshort probes (e.g., usually 16 nucleotides or longer for PCR orsequencing, and 40 nucleotides or longer for in situ hybridization). Thehigh stringency conditions used in these techniques are well known tothose skilled in the art of molecular biology, and examples of them canbe found, for example, in Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, N.Y., 1998, which is herebyincorporated by reference.

[0032] By “sample” is meant a tissue biopsy, amniotic fluid, cell,blood, serum, urine, stool, or other specimen obtained from a patient ora test subject. The sample can be analyzed to detect a mutation in aheart of glass gene, or expression levels of a heart of glass gene, bymethods that are known in the art. For example, methods such assequencing, single-strand conformational polymorphism (SSCP) analysis,or restriction fragment length polymorphism (RFLP) analysis of PCRproducts derived from a patient sample can be used to detect a mutationin a heart of glass gene; ELISA can be used to measure levels of a heartof glass polypeptide; and PCR can be used to measure the level of aheart of glass nucleic acid molecule.

[0033] By “heart of glass-related disease” or “heart of glass-relatedcondition” is meant a disease or condition that results frominappropriately high or low expression of a heart of glass gene, or amutation in a heart of glass gene that alters the biological activity ofa heart of glass nucleic acid molecule or polypeptide. Heart ofglass-related diseases and conditions can arise in any tissue in whichheart of glass is expressed during prenatal or post-natal life. Heart ofglass-related diseases and conditions can include heart diseases, suchas heart failure.

[0034] The invention provides several advantages. For example, using thediagnostic methods of the invention it is possible to detect anincreased likelihood of heart disease, such as heart failure, in apatient, so that appropriate intervention can be instituted before anysymptoms occur. This may be useful, for example, with patients inhigh-risk groups for heart failure. Also, the diagnostic methods of theinvention facilitate determination of the etiology of an existing heartcondition, such as heart failure, in a patient so that an appropriateapproach to treatment can be selected. In addition, the screeningmethods of the invention can be used to identify compounds that can beused to treat or to prevent heart conditions, such as heart failure.

[0035] Other features and advantages of the invention will be apparentfrom the following detailed description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036]FIG. 1 is a schematic representation of a map of the heg interval.YAC clones are indicated by the addresses beginning with “b.” The BACclone 110e08 contains recombinants on either side of the mutation andwas sequenced to determine the heg gene.

[0037]FIG. 2 is a schematic representation of the genomic structure ofthe heg gene. The gene comprises 22,000 basepairs of genomic sequence,not including the 5′ upstream region, and encodes an mRNA of 4.5 kb inlength.

[0038]FIG. 3 is a schematic representation of an alignment of the beggene with kiaa1237 (Genbank entry BAA86551.1). The cDNA sequence shownfor begI is derived from ligation of an oligo(dT)-primed 2.5 kb cDNAclone to a 2 kb 5′RACE product, and represents a putative secreted form.Heg2 is based on sequence of a PCR product using primers surrounding thestop codon in heg1 and encodes a potential membrane spanning domain anda highly conserved intracellular domain. The putative signal peptide andEGF-repeats are indicated with hashed lines. The premature stopassociated with the beg mutation is indicated by an asterisk.

DETAILED DESCRIPTION

[0039] The invention provides methods of diagnosing heart disease,screening methods for identifying compounds that can be used to treat orto prevent heart disease, and methods of treating or preventing heartdisease using such compounds. In particular, we have discovered that amutation (the heart of glass mutation) in a zebrafish gene, designatedherein as heart of glass, leads to a phenotype in zebrafish that issimilar to heart failure in mammals. Thus, the diagnostic methods of theinvention involve detection of mutations in genes encoding heart ofglass proteins, while the compound identification methods involvescreening for compounds that affect the phenotype of organisms havingmutations in genes encoding such proteins or other models of heartfailure. Compounds identified in this manner can be used in methods totreat or to prevent heart disease, such as heart failure.

[0040] The invention also provides animal model systems (e.g., zebrafishhaving mutations (e.g., the heart of glass mutation) in genes encodingthe heart of glass protein, or mice (or other animals) having suchmutations) that can be used in the screening methods mentioned above, aswell as the heart of glass protein, and genes encoding this protein.Also included in the invention are genes encoding mutant zebrafish heartof glass proteins (e.g., genes having the heart of glass mutation) andproteins encoded by these genes. Antibodies that specifically bind tothese proteins (wild type or mutant) are also included in the invention.

[0041] The diagnostic, screening, and therapeutic methods of theinvention, as well as the animal model systems, proteins, and genes ofthe invention, are described further, as follows.

[0042] Diagnostic Methods

[0043] Nucleic acid molecules encoding the heart of glass protein, aswell as polypeptides encoded by these nucleic acid molecules andantibodies specific for these polypeptides, can be used in methods todiagnose or to monitor diseases and conditions involving mutations in,or inappropriate expression of, genes encoding this protein. Asdiscussed above, the heart of glass mutation in zebrafish ischaracterized by a phenotype that is similar to that of heart failure inmammals, such as humans. Thus, detection of abnormalities in heart ofglass genes or in their expression can be used in methods to diagnose,or to monitor treatment or development of, human heart disease, such asheart failure.

[0044] The diagnostic methods of the invention can be used, for example,with patients that have heart failure, in an effort to determine itsetiology and, thus, to facilitate selection of an appropriate course oftreatment. The diagnostic methods can also be used with patients whohave not yet developed heart failure, but who are at risk of developingsuch a disease, or with patients that are at an early stage ofdeveloping such a disease. Also, the diagnostic methods of the inventioncan be used in prenatal genetic screening, for example, to identifyparents who may be carriers of a recessive mutation in a gene encoding aheart of glass protein.

[0045] Examples of heart failure that can be diagnosed (and treated)using the methods of the invention include congestive heart failure,which is characterized by fluid in the lungs or body, resulting fromfailure of the heart in acting as a pump; right sided heart failure(right ventricular), which is characterized by failure of the pumpingaction of the right ventricle, resulting in swelling of the body,especially the legs and abdomen; left sided heart failure (leftventricular), which is caused by failure of the pumping action of theleft side of the heart, resulting in congestion of the lungs; forwardheart failure, which is characterized by the inability of the heart topump blood forward at a sufficient rate to meet the oxygen needs of thebody at rest or during exercise; backward heart failure, which ischaracterized by the ability of the heart to meet the needs of the bodyonly if heart filling pressures are abnormally high; low-output, whichis characterized by failure to maintain blood output; and high-output,which is characterized by heart failure symptoms, even when cardiacoutput is high.

[0046] Heart of glass may also play a role in cardiovascular diseasesother than heart failure, such as coronary artery disease or conditionsassociated with valve formation defects, and, thus, detection ofabnormalities in heart of glass genes or their expression can be used inmethods to diagnose and monitor these conditions as well. The methods ofthe invention can be used to diagnose (or to treat) the disordersdescribed herein in any mammal, for example, humans, domestic pets, orlivestock.

[0047] Abnormalities in heart of glass that can be detected using thediagnostic methods of the invention include those characterized by, forexample, (i) a gene encoding a heart of glass protein containing amutation that results in the production of an abnormal heart of glassprotein, (ii) an abnormal heart of glass polypeptide itself, and (iii) amutation in a gene encoding a heart of glass protein that results inproduction of an abnormal amount of this protein. Detection of suchabnormalities can be used in methods to diagnose human heart disease,such as heart failure (see above). Exemplary of the mutations in a heartof glass protein is the heart of glass mutation, which is describedfurther below.

[0048] A mutation in a gene encoding a heart of glass protein can bedetected in any tissue of a subject, even one in which this protein isnot expressed. Because of the limited number of tissues in which theseproteins are expressed (e.g., the myocardium, neurons, and smoothmuscle) and because of the undesirability of sampling such tissues forassays, it may be preferable to detect mutant genes in other, moreeasily obtained sample types, such as in blood or amniotic fluidsamples.

[0049] Detection of a mutation in a gene encoding a heart of glassprotein can be carried out using any standard diagnostic technique. Forexample, a biological sample obtained from a patient can be analyzed forone or more mutations (e.g., a heart of glass mutation; see below) innucleic acid molecules encoding a heart of glass protein using amismatch detection approach. Generally, this approach involvespolymerase chain reaction (PCR) amplification of nucleic acid moleculesfrom a patient sample, followed by identification of a mutation (i.e., amismatch) by detection of altered hybridization, aberrantelectrophoretic gel migration, binding, or cleavage mediated by mismatchbinding proteins, or by direct nucleic acid molecule sequencing. Any ofthese techniques can be used to facilitate detection of a mutant geneencoding a heart of glass protein, and each is well known in the art.For instance, examples of these techniques are described by Orita et al.(Proc. Natl. Acad. Sci. U.S.A. 86:2766-2770, 1989) and Sheffield et al.(Proc. Natl. Acad. Sci. U.S.A. 86:232-236, 1989).

[0050] In addition to facilitating diagnosis of existing heart disease,mutation detection assays also provide an opportunity to diagnose apredisposition to heart disease related to a mutation in a gene encodinga heart of glass protein before the onset of symptoms. For example, apatient who is heterozygous for a gene encoding an abnormal heart ofglass protein (or an abnormal amount thereof) that suppresses normalheart of glass biological activity or expression may show no clinicalsymptoms of a disease related to such proteins, and yet possess a higherthan normal probability of developing heart disease, such as heartfailure. Given such a diagnosis, a patient can take precautions tominimize exposure to adverse environmental factors, and can carefullymonitor their medical condition, for example, through frequent physicalexaminations. As mentioned above, this type of diagnostic approach canalso be used to detect a mutation in a gene encoding the heart of glassprotein in prenatal screens.

[0051] While it may be preferable to carry out diagnostic methods fordetecting a mutation in a gene encoding the heart of glass protein usinggenomic DNA from readily accessible tissues, mRNA encoding this protein,or the protein itself, can also be assayed from tissue samples in whichit is expressed, and may not be so readily accessible. For example,expression levels of a gene encoding the heart of glass protein in sucha tissue sample from a patient can be determined by using any of anumber of standard techniques that are well known in the art, includingnorthern blot analysis and quantitative PCR (see, e.g., Ausubel et al.,supra; PCR Technology: Principles and Applications for DNAAmplification, H. A. Ehrlich, Ed., Stockton Press, NY; Yap et al. Nucl.Acids. Res. 19:4294, 1991).

[0052] In another diagnostic approach of the invention, an immunoassayis used to detect or to monitor the level of a heart of glass protein ina biological sample. Polyclonal or monoclonal antibodies specific forthe heart of glass protein can be used in any standard immunoassayformat (e.g., ELISA, Western blot, or RIA; see, e.g., Ausubel et al.,supra) to measure polypeptide levels of the heart of glass protein.These levels can be compared to levels of the heart of glass protein ina sample from an unaffected individual. Detection of a decrease inproduction of the heart of glass protein using this method, for example,may be indicative of a condition or a predisposition to a conditioninvolving insufficient biological activity of the heart of glassprotein.

[0053] Immunohistochemical techniques can also be utilized for detectionof the heart of glass protein in patient samples. For example, a tissuesample can be obtained from a patient, sectioned, and stained for thepresence of the heart of glass protein using an anti-heart of glassprotein antibody and any standard detection system (e.g., one thatincludes a secondary antibody conjugated to an enzyme such ashorseradish peroxidase). General guidance regarding such techniques canbe found in, e.g., Bancroft et al., Theory and Practice of HistologicalTechniques, Churchill Livingstone, 1982, and Ausubel et al., supra.

[0054] Identification of Molecules that can be used to Treat or toPrevent Heart Failure

[0055] Identification of a mutation in the heart of glass gene encodingthe heart of glass protein as resulting in a phenotype that is relatedto heart failure facilitates the identification of molecules (e.g.,small organic or inorganic molecules, peptides, or nucleic acidmolecules) that can be used to treat or to prevent heart disease, suchas heart failure. The effects of candidate compounds on heart failurecan be investigated using, for example, the zebrafish system. Thezebrafish, Danio rerio, is a convenient organism to use in geneticanalysis of vascular development. In addition to its short generationtime and fecundity, it has an accessible and transparent embryo,allowing direct observation of blood vessel function from the earlieststages of development. As discussed further below, zebrafish and otheranimals having a heart of glass mutation, which can be used in thesemethods, are also included in the invention.

[0056] In one example of the screening methods of the invention, azebrafish having a mutation in a gene encoding the heart of glassprotein (e.g, a zebrafish having the heart of glass mutation) iscontacted with a candidate compound, and the effect of the compound onthe development of a heart abnormality that is characteristic of heartfailure, or on the status of such an existing heart abnormality, ismonitored relative to an untreated, identically mutant control. Asdiscussed further below, zebrafish having the heart of glass mutationare characterized by an enlarged and distended heart. Thus, thesecharacteristics (in addition to other characteristics of heart disease)can be monitored using the screening methods of the invention.

[0057] After a compound has been shown to have a desired effect in thezebrafish system, it can be tested in other models of heart disease, forexample, in mice or other animals having a mutation in a gene encodingthe heart of glass protein. Alternatively, testing in such animal modelsystems can be carried out in the absence of zebrafish testing.

[0058] Cell culture-based assays can also be used in the identificationof molecules that increase or decrease heart of glass levels orbiological activity. According to one approach, candidate molecules areadded at varying concentrations to the culture medium of cellsexpressing heart of glass mRNA. Heart of glass biological activity isthen measured using standard techniques. The measurement of biologicalactivity can include the measurement of heart of glass protein andnucleic acid molecule levels.

[0059] In general, novel drugs for prevention or treatment of heartdiseases related to mutations in a gene encoding the heart of glassprotein can be identified from large libraries of natural products,synthetic (or semi-synthetic) extracts, and chemical libraries usingmethods that are well known in the art. Those skilled in the field ofdrug discovery and development will understand that the precise sourceof test extracts or compounds is not critical to the screening methodsof the invention and that dereplication, or the elimination ofreplicates or repeats of materials already known for their therapeuticactivities for heart disease can be employed whenever possible.

[0060] Candidate compounds to be tested include purified (orsubstantially purified) molecules or one or more component of a mixtureof compounds (e.g., an extract or supernatant obtained from cells;Ausubel et al., supra) and such compounds further include both naturallyoccurring or artificially derived chemicals and modifications ofexisting compounds. For example, candidate compounds can bepolypeptides, synthesized organic or inorganic molecules, naturallyoccurring organic or inorganic molecules, nucleic acid molecules, andcomponents thereof.

[0061] Numerous sources of naturally occurring candidate compounds arereadily available to those skilled in the art. For example, naturallyoccurring compounds can be found in cell (including plant, fungal,prokaryotic, and animal) extracts, mammalian serum, growth medium inwhich mammalian cells have been cultured, protein expression libraries,or fermentation broths. In addition, libraries of natural compounds inthe form of bacterial, fungal, plant, and animal extracts arecommercially available from a number of sources, including Biotics(Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceanographic Institute(Ft. Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.).Furthermore, libraries of natural compounds can be produced, if desired,according to methods that are known in the art, e.g., by standardextraction and fractionation.

[0062] Artificially derived candidate compounds are also readilyavailable to those skilled in the art. Numerous methods are availablefor generating random or directed synthesis (e.g., semi-synthesis ortotal synthesis) of any number of chemical compounds, including, forexample, saccharide-, lipid-, peptide-, and nucleic acid molecule-basedcompounds. In addition, synthetic compound libraries are commerciallyavailable from Brandon Associates (Merrimack, N.H.) and AldrichChemicals (Milwaukee, Wis.). Libraries of synthetic compounds can alsobe produced, if desired, according to methods known in the art, e.g., bystandard extraction and fractionation. Furthermore, if desired, anylibrary or compound can be readily modified using standard chemical,physical, or biochemical methods.

[0063] When a crude extract is found to have an effect on thedevelopment or persistence of heart disease, further fractionation ofthe positive lead extract can be carried out to isolate chemicalconstituents responsible for the observed effect. Thus, the goal of theextraction, fractionation, and purification process is the carefulcharacterization and identification of a chemical entity within thecrude extract having a desired activity. The same assays describedherein for the detection of activities in mixtures of compounds can beused to purify the active component and to test derivatives of thesecompounds. Methods of fractionation and purification of suchheterogeneous extracts are well known in the art. If desired, compoundsshown to be useful agents for treatment can be chemically modifiedaccording to methods known in the art.

[0064] Animal Model Systems

[0065] The invention also provides animal model systems for use incarrying out the screening methods described above. Examples of thesemodel systems include zebrafish and other animals, such as mice, thathave a mutation (e.g., the heart of glass mutation) in a gene encodingthe heart of glass protein. For example, a zebrafish model that can beused in the invention can include a mutation that results in a lack ofheart of glass protein production or production of a truncated (e.g., byintroduction of a stop codon) or otherwise altered heart of glass geneproduct. As a specific example, a zebrafish having the heart of glassmutation can be used (see below).

[0066] Treatment or Prevention of Heart Failure

[0067] Compounds identified using the screening methods described abovecan be used to treat patients that have or are at risk of developingheart disease, such as heart failure. Nucleic acid molecules encodingthe heart of glass protein, as well as these proteins themselves, canalso be used in such methods. Treatment may be required only for a shortperiod of time or may, in some form, be required throughout a patient'slifetime. Any continued need for treatment, however, can be determinedusing, for example, the diagnostic methods described above. Inconsidering various therapies, it is to be understood that suchtherapies are, preferably, targeted to the affected or potentiallyaffected organ (i.e., the heart).

[0068] Treatment or prevention of diseases resulting from a mutated geneencoding the heart of glass protein can be accomplished, for example, bymodulating the function of a mutant heart of glass protein. Treatmentcan also be accomplished by delivering normal heart of glass protein toappropriate cells, altering the levels of normal or mutant heart ofglass protein, replacing a mutant gene encoding a heart of glass proteinwith a normal gene encoding the heart of glass protein, or administeringa normal gene encoding the heart of glass protein. It is also possibleto correct the effects of a defect in a gene encoding the heart of glassprotein by modifying the physiological pathway (e.g., a signaltransduction pathway) in which the heart of glass protein participates.

[0069] In a patient diagnosed as being heterozygous for a gene encodinga mutant heart of glass protein, or as susceptible to such mutations oraberrant heart of glass expression (even if those mutations orexpression patterns do not yet result in alterations in expression orbiological activity of the heart of glass), any of the therapiesdescribed herein can be administered before the occurrence of thedisease phenotype. In particular, compounds shown to have an effect onthe phenotype of mutants, or to modulate expression of heart of glassproteins can be administered to patients diagnosed with potential oractual heart disease by any standard dosage and route of administration.

[0070] Any appropriate route of administration can be employed toadminister a compound found to be effective in treating or preventingheart failure according to the invention. For example, administrationcan be parenteral, intravenous, intra-arterial, subcutaneous,intramuscular, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, by aerosol, by suppository,or oral.

[0071] A therapeutic compound of the invention can be administeredwithin a pharmaceutically-acceptable diluent, carrier, or excipient, inunit dosage form. Administration can begin before or after the patientis symptomatic. Methods that are well known in the art for makingformulations are found, for example, in Remington 's PharmaceuticalSciences (18^(th) edition), ed. A. Gennaro, 1990, Mack PublishingCompany, Easton, Pa. Therapeutic formulations can be in the form ofliquid solutions or suspensions. Formulations for parenteraladministration can, for example, contain excipients; sterile water; orsaline; polyalkylene glycols, such as polyethylene glycol; oils ofvegetable origin; or hydrogenated napthalenes. Biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers can be used to control therelease of the compounds. Other potentially useful parenteral deliverysystems for compounds identified using the methods of the inventioninclude ethylene-vinyl acetate copolymer particles, osmotic pumps,implantable infusion systems, and liposomes. For oral administration,formulations can be in the form of tablets or capsules. Formulations forinhalation can contain excipients, for example, lactose, or can beaqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate, and deoxycholate, or can be oily solutions foradministration in the form of nasal drops, or as a gel. Alternatively,intranasal formulations can be in the form of powders or aerosols.

[0072] To replace a mutant protein with normal protein, or to addprotein to cells that do not express sufficient or normal heart of glassprotein, it may be necessary to obtain large amounts of pure heart ofglass protein from cultured cell systems in which the protein isexpressed (see, e.g., below). Delivery of the protein to the affectedtissue can then be accomplished using appropriate packaging oradministration systems.

[0073] Gene therapy is another therapeutic approach for preventing orameliorating diseases (e.g., heart failure) caused by heart of glassgene defects. Nucleic acid molecules encoding wild type heart of glassprotein can be delivered to cells that lack sufficient, normal heart ofglass protein biological activity (e.g., cells carrying mutations (e.g.,the heart of glass mutation) in heart of glass genes). The nucleic acidmolecules must be delivered to those cells in a form in which they canbe taken up by the cells and so that sufficient levels of protein, toprovide effective heart of glass protein function, can be produced.Alternatively, for some heart of glass mutations, it may be possibleslow the progression of the resulting disease or to modulate heart ofglass protein activity by introducing another copy of a homologous genebearing a second mutation in that gene, to alter the mutation, or to useanother gene to block any negative effect.

[0074] Transducing retroviral, adenoviral, and adeno-associated viralvectors can be used for somatic cell gene therapy, especially because oftheir high efficiency of infection and stable integration and expression(see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kidoet al., Current Eye Research 15:833-844,1996; Bloomer et al., Journal ofVirology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996;and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). Forexample, the full length heart of glass gene, or a portion thereof, canbe cloned into a retroviral vector and expression can be driven from itsendogenous promoter, from the retroviral long terminal repeat, or from apromoter specific for a target cell type of interest (such as cardiacmuscle or other vascular cells). Other viral vectors that can be usedinclude, for example, vaccinia virus, bovine papilloma virus, or aherpes virus, such as Epstein-Barr Virus (also see, for example, thevectors of Miller, Human Gene Therapy 15-14,1990; Friedman, Science244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988;Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990;Sharp, The Lancet 337:1277-1278, 1991; Cometta et al., Nucleic AcidResearch and Molecular Biology 36:311-322, 1987; Anderson, Science226:401-409,1984; Moen, Blood Cells 17:407-416, 1991; Miller et al.,Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviralvectors are particularly well developed and have been used in clinicalsettings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson etal., U.S. Pat. No. 5,399,346).

[0075] Non-viral approaches can also be employed for the introduction oftherapeutic DNA into cells predicted to be subject to diseases involvingthe heart of glass protein. For example, a heart of glass nucleic acidmolecule or an antisense nucleic acid molecule can be introduced into acell by lipofection (Felgner et al., Proc. Natl. Acad. Sci. U.S.A.84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham etal., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods inEnzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wuet al., Journal of Biological Chemistry 263:14621, 1988; Wu et al.,Journal of Biological Chemistry 264:16985, 1989), or by micro-injectionunder surgical conditions (Wolff et al., Science 247:1465, 1990).

[0076] Gene transfer can also be achieved using non-viral meansinvolving transfection in vitro. Such methods include use of calciumphosphate, DEAE dextran, electroporation, and protoplast fusion.Liposomes can also be potentially beneficial for delivery of DNA into acell. Transplantation of normal genes into the affected tissues of apatient can also be accomplished by transferring a normal heart of glassprotein into a cultivatable cell type ex vivo, after which the cell (orits descendants) are injected into a targeted tissue.

[0077] Heart of glass cDNA expression for use in gene therapy methodscan be directed from any suitable promoter (e.g., the humancytomegalovirus (CMV), simian virus 40 (SV40), or metallothioneinpromoters), and regulated by any appropriate mammalian regulatoryelement. For example, if desired, enhancers known to preferentiallydirect gene expression in specific cell types can be used to directheart of glass expression. The enhancers used can include, withoutlimitation, those that are characterized as tissue- or cell-specificenhancers. Alternatively, if a heart of glass genomic clone is used as atherapeutic construct (such clones can be identified by hybridizationwith heart of glass cDNA, as described herein), regulation can bemediated by the cognate regulatory sequences or, if desired, byregulatory sequences derived from a heterologous source, including anyof the promoters or regulatory elements described above.

[0078] Antisense-based strategies can be employed to explore heart ofglass protein gene function and as a basis for therapeutic drug design.These strategies are based on the principle that sequence-specificsuppression of gene expression (via transcription or translation) can beachieved by intracellular hybridization between genomic DNA or mRNA anda complementary antisense species. The formation of a hybrid RNA duplexinterferes with transcription of the target heart of glassprotein-encoding genomic DNA molecule, or processing, transport,translation, or stability of the target heart of glass mRNA molecule.

[0079] Antisense strategies can be delivered by a variety of approaches.For example, antisense oligonucleotides or antisense RNA can be directlyadministered (e.g., by intravenous injection) to a subject in a formthat allows uptake into cells. Alternatively, viral or plasmid vectorsthat encode antisense RNA (or antisense RNA fragments) can be introducedinto a cell in vivo or ex vivo. Antisense effects can be induced bycontrol (sense) sequences; however, the extent of phenotypic changes ishighly variable. Phenotypic effects induced by antisense effects arebased on changes in criteria such as protein levels, protein activitymeasurement, and target mRNA levels.

[0080] Heart of glass gene therapy can also be accomplished by directadministration of antisense heart of glass mRNA to a cell that isexpected to be adversely affected by the expression of wild type ormutant heart of glass protein. The antisense heart of glass mRNA can beproduced and isolated by any standard technique, but is most readilyproduced by in vitro transcription using an antisense heart of glasscDNA under the control of a high efficiency promoter (e.g., the T7promoter). Administration of antisense heart of glass mRNA to cells canbe carried out by any of the methods for direct nucleic acid moleculeadministration described above.

[0081] An alternative strategy for inhibiting heart of glass proteinfunction using gene therapy involves intracellular expression of ananti-heart of glass protein antibody or a portion of an anti-heart ofglass protein antibody. For example, the gene (or gene fragment)encoding a monoclonal antibody that specifically binds to a heart ofglass protein and inhibits its biological activity can be placed underthe transcriptional control of a tissue-specific gene regulatorysequence.

[0082] Another therapeutic approach included in the invention involvesadministration of a recombinant heart of glass polypeptide, eitherdirectly to the site of a potential or actual disease-affected tissue(for example, by injection) or systemically (for example, by anyconventional recombinant protein administration technique). The dosageof the heart of glass protein depends on a number of factors, includingthe size and health of the individual patient but, generally, between0.1 mg and 100 mg, inclusive, is administered per day to an adult in anypharmaceutically acceptable formulation.

[0083] Synthesis of Heart of Glass Proteins, Polypeptides, andPolypeptide Fragments

[0084] Those skilled in the art of molecular biology will understandthat a wide variety of expression systems can be used to produce therecombinant heart of glass proteins. As discussed further below, theprecise host cell used is not critical to the invention. The heart ofglass proteins can be produced in a prokaryotic host (e.g., E. coli) orin a eukaryotic host (e.g., S. cerevisiae, insect cells such as Sf9cells, or mammalian cells such as COS-1, NIH 3T3, or HeLa cells). Thesecells are commercially available from, for example, the American TypeCulture Collection, Manassas, Va. (see also Ausubel et al., supra). Themethod of transformation and the choice of expression vehicle (e.g.,expression vector) will depend on the host system selected.Transformation and transfection methods are described, e.g., in Ausubelet al., supra, and expression vehicles can be chosen from thoseprovided, e.g., in Pouwels et al., Cloning Vectors: A Laboratory Manual,1985, Supp. 1987. Specific examples of expression systems that can beused in the invention are described further, as follows.

[0085] For protein expression, eukaryotic or prokaryotic expressionsystems can be generated in which heart of glass gene sequences areintroduced into a plasmid or other vector, which is then used totransform living cells. Constructs in which full-length heart of glasscDNAs, containing the entire open reading frame, inserted in the correctorientation into an expression plasmid can be used for proteinexpression. Alternatively, portions of heart of glass gene sequences,including wild type or mutant heart of glass sequences, can be inserted.Prokaryotic and eukaryotic expression systems allow various importantfunctional domains of heart of glass proteins to be recovered, ifdesired, as fusion proteins, and then used for binding, structural, andfunctional studies, and also for the generation of antibodies.

[0086] Typical expression vectors contain promoters that directsynthesis of large amounts of mRNA corresponding to a nucleic acidmolecule that has been inserted into the vector. They can also include aeukaryotic or prokaryotic origin of replication, allowing for autonomousreplication within a host cell, sequences that confer resistance to anotherwise toxic drug, thus allowing vector-containing cells to beselected in the presence of the drug, and sequences that increase theefficiency with which the synthesized mRNA is translated. Stablelong-term vectors can be maintained as freely replicating entities byusing regulatory elements of, for example, viruses (e.g., the OriPsequences from the Epstein Barr Virus genome). Cell lines can also beproduced that have the vector integrated into genomic DNA of the cells,and, in this manner, the gene product can be produced in the cells on acontinuous basis.

[0087] Expression of foreign molecules in bacteria, such as Escherichiacoli, requires the insertion of a foreign nucleic acid molecule, e.g., aheart of glass nucleic acid molecule, into a bacterial expressionvector. Such plasmid vectors include several elements required for thepropagation of the plasmid in bacteria, and for expression of foreignDNA contained within the plasmid. Propagation of only plasmid-bearingbacteria is achieved by introducing, into the plasmid, a selectablemarker-encoding gene that allows plasmid-bearing bacteria to grow in thepresence of an otherwise toxic drug. The plasmid also contains atranscriptional promoter capable of directing synthesis of large amountsof mRNA from the foreign DNA. Such promoters can be, but are notnecessarily, inducible promoters that initiate transcription uponinduction by culture under appropriate conditions (e.g., in the presenceof a drug that activates the promoter). The plasmid also, preferably,contains a polylinker to simplify insertion of the gene in the correctorientation within the vector.

[0088] Once an appropriate expression vector containing a heart of glassgene, or a fragment, fusion, or mutant thereof, is constructed, it canbe introduced into an appropriate host cell using a transformationtechnique, such as, for example, calcium phosphate transfection,DEAE-dextran transfection, electroporation, microinjection, protoplastfusion, or liposome-mediated transfection. Host cells that can betransfected with the vectors of this invention can include, but are notlimited to, E. coli or other bacteria, yeast, fungi, insect cells(using, for example, baculoviral vectors for expression), or cellsderived from mice, humans, or other animals. Mammalian cells can also beused to express heart of glass proteins using a virus expression system(e.g., a vaccinia virus expression system) described, for example, inAusubel et al., supra.

[0089] In vitro expression of heart of glass proteins, fusions,polypeptide fragments, or mutants encoded by cloned DNA can also becarried out using the T7 late-promoter expression system. This systemdepends on the regulated expression of T7 RNA polymerase, an enzymeencoded in the DNA of bacteriophage T7. The T7 RNA polymerase initiatestranscription at a specific 23-bp promoter sequence called the T7 latepromoter. Copies of the T7 late promoter are located at several sites onthe T7 genome, but none are present in E. coli chromosomal DNA. As aresult, in T7-infected E. coli, T7 RNA polymerase catalyzestranscription of viral genes, but not E. coli genes. In this expressionsystem, recombinant E. coli cells are first engineered to carry the geneencoding T7 RNA polymerase next to the lac promoter. In the presence ofIPTG, these cells transcribe the T7 polymerase gene at a high rate andsynthesize abundant amounts of T7 RNA polymerase. These cells are thentransformed with plasmid vectors that carry a copy of the T7 latepromoter protein. When IPTG is added to the culture medium containingthese transformed E. coli cells, large amounts of T7 RNA polymerase areproduced. The polymerase then binds to the T7 late promoter on theplasmid expression vectors, catalyzing transcription of the insertedcDNA at a high rate. Since each E. coli cell contains many copies of theexpression vector, large amounts of mRNA corresponding to the clonedcDNA can be produced in this system and the resulting protein can beradioactively labeled.

[0090] Plasmid vectors containing late promoters and the correspondingRNA polymerases from related bacteriophages, such as T3, T5, and SP6,can also be used for in vitro production of proteins from cloned DNA. E.coli can also be used for expression using an M13 phage, such as mGPI-2.Furthermore, vectors that contain phage lambda regulatory sequences, orvectors that direct the expression of fusion proteins, for example, amaltose-binding protein fusion protein or a glutathione-S-transferasefusion protein, also can be used for expression in E. coli.

[0091] Eukaryotic expression systems are useful for obtainingappropriate post-translational modification of expressed proteins.Transient transfection of a eukaryotic expression plasmid containing aheart of glass protein into a eukaryotic host cell allows the transientproduction of a heart of glass protein by the transfected host cell.Heart of glass proteins can also be produced by a stably-transfectedeukaryotic (e.g., mammalian) cell line. A number of vectors suitable forstable transfection of mammalian cells are available to the public (see,e.g., Pouwels et al., supra), as are methods for constructing linesincluding such cells (see, e.g., Ausubel et al., supra).

[0092] In one example, cDNA encoding a heart of glass protein, fusion,mutant, or polypeptide fragment is cloned into an expression vector thatincludes the dihydrofolate reductase (DHFR) gene. Integration of theplasmid and, therefore, integration of the heart of glassprotein-encoding gene, into the host cell chromosome is selected for byinclusion of 0.01-300 μM methotrexate in the cell culture medium(Ausubel et al., supra). This dominant selection can be accomplished inmost cell types. Recombinant protein expression can be increased byDHFR-mediated amplification of the transfected gene. Methods forselecting cell lines bearing gene amplifications are described inAusubel et al., supra. These methods generally involve extended culturein medium containing gradually increasing levels of methotrexate. Themost commonly used DHFR-containing expression vectors are pCVSEII-DHFRand pAdD26SV(A) (described, for example, in Ausubel et al., supra). Thehost cells described above or, preferably, a DHFR-deficient CHO cellline (e.g., CHO DHFR-cells, ATCC Accession No. CRL 9096) are among thosethat are most preferred for DHFR selection of a stably transfected cellline or DHFR-mediated gene amplification.

[0093] Another preferred eukaryotic expression system is the baculovirussystem using, for example, the vector pBacPAK9, which is available fromClontech (Palo Alto, Calif.). If desired, this system can be used inconjunction with other protein expression techniques, for example, themyc tag approach described by Evan et al. (Molecular and CellularBiology 5:3610-3616, 1985).

[0094] Once a recombinant protein is expressed, it can be isolated fromthe expressing cells by cell lysis followed by protein purificationtechniques, such as affinity chromatography. In this example, ananti-heart of glass protein antibody, which can be produced by themethods described herein, can be attached to a column and used toisolate the recombinant heart of glass proteins. Lysis and fractionationof heart of glass protein-harboring cells prior to affinitychromatography can be performed by standard methods (see, e.g., Ausubelet al., supra). Once isolated, the recombinant protein can, if desired,be purified further by, e.g., high performance liquid chromatography(HPLC; e.g., see Fisher, Laboratory Techniques In Biochemistry andMolecular Biology, Work and Burdon, Eds., Elsevier, 1980).

[0095] Polypeptides of the invention, particularly short heart of glassprotein fragments and longer fragments of the N-terminus and C-terminusof the heart of glass protein, can also be produced by chemicalsynthesis (e.g., by the methods described in Solid Phase PeptideSynthesis, 2^(nd) ed., 1984, The Pierce Chemical Co., Rockford, Ill.).These general techniques of polypeptide expression and purification canalso be used to produce and isolate useful heart of glass proteinfragments or analogs, as described herein.

[0096] Heart of Glass Protein Fragments

[0097] Polypeptide fragments that include various portions of heart ofglass proteins are useful in identifying the domains of the heart ofglass protein that are important for its biological activities, such asprotein-protein interactions and transcription. Methods for generatingsuch fragments are well known in the art (see, for example, Ausubel etal., supra), using the nucleotide sequences provided herein. Forexample, a heart of glass protein fragment can be generated by PCRamplifying a desired heart of glass protein nucleic acid moleculefragment using oligonucleotide primers designed based upon heart ofglass nucleic acid sequences. Preferably, the oligonucleotide primersinclude unique restriction enzyme sites that facilitate insertion of theamplified fragment into the cloning site of an expression vector (e.g.,a mammalian expression vector, see above). This vector can then beintroduced into a cell (e.g., a mammalian cell; see above) by artifice,using any of the various techniques that are known in the art, such asthose described herein, resulting in the production of a heart of glassprotein fragment in the cell containing the expression vector. Heart ofglass protein fragments (e.g., chimeric fusion proteins) can also beused to raise antibodies specific for various regions of the heart ofglass protein using, for example, the methods described below.

[0098] Heart of Glass Protein Antibodies

[0099] To prepare polyclonal antibodies, heart of glass proteins,fragments of heart of glass proteins, or fusion proteins containingdefined portions of heart of glass proteins can be synthesized in, e.g.,bacteria by expression of corresponding DNA sequences contained in asuitable cloning vehicle. Fusion proteins are commonly used as a sourceof antigen for producing antibodies. Two widely used expression systemsfor E. coli are lacZ fusions using the pUR series of vectors and trpEfusions using the pATH vectors. The proteins can be purified, coupled toa carrier protein, mixed with Freund's adjuvant to enhance stimulationof the antigenic response in an inoculated animal, and injected intorabbits or other laboratory animals. Alternatively, protein can beisolated from heart of glass protein-expressing cultured cells.Following booster injections at bi-weekly intervals, the rabbits orother laboratory animals are then bled and the sera isolated. The seracan be used directly or can be purified prior to use by various methods,including affinity chromatography employing reagents such as ProteinA-Sepharose, antigen-Sepharose, and anti-mouse-Ig-Sepharose. The seracan then be used to probe protein extracts from heart of glassprotein-expressing tissue fractionated by polyacrylamide gelelectrophoresis to identify heart of glass proteins. Alternatively,synthetic peptides can be made that correspond to antigenic portions ofthe protein and used to inoculate the animals.

[0100] To generate peptide or full-length protein for use in making, forexample, heart of glass protein-specific antibodies, a heart of glassprotein coding sequence can be expressed as a C-terminal or N-terminalfusion with glutathione S-transferase (GST; Smith et al., Gene 67:3140,1988). The fusion protein can be purified on glutathione-Sepharosebeads, eluted with glutathione, cleaved with a protease, such asthrombin or Factor-Xa (at the engineered cleavage site), and purified tothe degree required to successfully immunize rabbits. Primaryimmunizations can be carried out with Freund's complete adjuvant andsubsequent immunizations performed with Freund's incomplete adjuvant.Antibody titers can be monitored by Western blot and immunoprecipitationanalyses using the protcase-cleaved heart of glass protein fragment ofthe GST-heart of glass protein. Immune sera can be affinity purifiedusing CNBr—Sepharose-coupled heart of glass protein. Antiserumspecificity can be determined using a panel of unrelated GST fusionproteins.

[0101] Alternatively, monoclonal heart of glass protein antibodies canbe produced by using, as an antigen, heart of glass protein isolatedfrom heart of glass protein-expressing cultured cells or heart of glassprotein isolated from tissues. The cell extracts, or recombinant proteinextracts containing heart of glass protein, can, for example, beinjected with Freund's adjuvant into mice. Several days after beinginjected, the mouse spleens can be removed, the tissues disaggregated,and the spleen cells suspended in phosphate buffered saline (PBS). Thespleen cells serve as a source of lymphocytes, some of which would beproducing antibody of the appropriate specificity. These can then befused with permanently growing myeloma partner cells, and the productsof the fusion plated into a number of tissue culture wells in thepresence of selective agents, such as hypoxanthine, aminopterine, andthymidine (HAT). The wells can then be screened by ELISA to identifythose containing cells making antibody capable of binding to a heart ofglass protein, polypeptide fragment, or mutant thereof. These cells canthen be re-plated and, after a period of growth, the wells containingthese cells can be screened again to identify antibody-producing cells.Several cloning procedures can be carried out until over 90% of thewells contain single clones that are positive for specific antibodyproduction. From this procedure, a stable line of clones that producethe antibody can be established. The monoclonal antibody can then bepurified by affinity chromatography using Protein A Sepharose and ionexchange chromatography, as well as variations and combinations of thesetechniques. Once produced, monoclonal antibodies are also tested forspecific heart of glass protein recognition by Western blot orimmunoprecipitation analysis (see, e.g., Kohler et al., Nature 256:495,1975; Kohler et al., European Journal of Immunology 6:511, 1976; Kohleret al., European Journal of Immunology 6:292, 1976; Hammerling et al.,In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, New York,N.Y., 1981; Ausubel et al., supra).

[0102] As an alternate or adjunct immunogen to GST fusion proteins,peptides corresponding to relatively unique hydrophilic regions of theheart of glass protein can be generated and coupled to keyhole limpethemocyanin (KLH) through an introduced C-terminal lysine. Antiserum toeach of these peptides can be similarly affinity-purified on peptidesconjugated to BSA, and specificity tested by ELISA and Western blottingusing peptide conjugates, and by Western blotting andimmunoprecipitation using the heart of glass protein, for example,expressed as a GST fusion protein.

[0103] Antibodies of the invention can be produced using heart of glassprotein amino acid sequences that do not reside within highly conservedregions, and that appear likely to be antigenic, as analyzed by criteriasuch as those provided by the Peptide Structure Program (GeneticsComputer Group Sequence Analysis Package, Program Manual for the GCGPackage, Version 7, 1991) using the algorithm of Jameson et al., CABIOS4:181, 1988. These fragments can be generated by standard techniques,e.g., by PCR, and cloned into the pGEX expression vector. GST fusionproteins can be expressed in E. coli and purified using aglutathione-agarose affinity matrix (Ausubel et al., supra). To generaterabbit polyclonal antibodies, and to minimize the potential forobtaining antisera that is non-specific, or exhibits low-affinitybinding to a heart of glass protein, two or three fusions are generatedfor each protein, and each fusion is injected into at least two rabbits.Antisera are raised by injections in series, preferably including atleast three booster injections.

[0104] In addition to intact monoclonal and polyclonal anti-heart ofglass protein antibodies, the invention features various geneticallyengineered antibodies, humanized antibodies, and antibody fragments,including F(ab′)2, Fab′, Fab, Fv, and sFv fragments. Truncated versionsof monoclonal antibodies, for example, can be produced by recombinantmethods in which plasmids are generated that express the desiredmonoclonal antibody fragment(s) in a suitable host. Antibodies can behumanized by methods known in the art, e.g., monoclonal antibodies witha desired binding specificity can be commercially humanized (Scotgene,Scotland; Oxford Molecular, Palo Alto, Calif.). Fully human antibodies,such as those expressed in transgenic animals, are also included in theinvention (Green et al., Nature Genetics 7:13-21, 1994).

[0105] Ladner (U.S. Pat. Nos. 4,946,778 and 4,704,692) describes methodsfor preparing single polypeptide chain antibodies. Ward et al., Nature341:544-546, 1989, describes the preparation of heavy chain variabledomains, which they term “single domain antibodies,” and which have highantigen-binding affinities. McCafferty et al., Nature 348:552-554, 1990,show that complete antibody V domains can be displayed on the surface offd bacteriophage, that the phage bind specifically to antigen, and thatrare phage (one in a million) can be isolated after affinitychromatography. Boss et al., U.S. Pat. No. 4,816,397, describes variousmethods for producing immunoglobulins, and immunologically functionalfragments thereof, that include at least the variable domains of theheavy and light chains in a single host cell. Cabilly et al., U.S. Pat.No. 4,816,567, describes methods for preparing chimeric antibodies.

[0106] Use of Heart of Glass Antibodies

[0107] Antibodies to heart of glass proteins can be used, as notedabove, to detect heart of glass proteins or to inhibit the biologicalactivities of heart of glass proteins. For example, a nucleic acidmolecule encoding an antibody or portion of an antibody can be expressedwithin a cell to inhibit heart of glass protein function. In addition,the antibodies can be coupled to compounds, such as radionuclides andliposomes, for diagnostic or therapeutic uses. Antibodies thatspecifically recognize extracellular domains of heart of glass proteinsare useful for targeting such attached moieties to cells displaying suchheart of glass protein domains at their surfaces. Antibodies thatinhibit the activity of a heart of glass polypeptide described hereincan also be useful in preventing or slowing the development of a diseasecaused by inappropriate expression of a wild type or mutant heart ofglass gene.

[0108] Detection of Heart of Glass Gene Expression

[0109] As noted, the antibodies described above can be used to monitorheart of glass protein expression. In situ hybridization of RNA can beused to detect the expression of heart of glass genes. RNA in situhybridization techniques rely upon the hybridization of a specificallylabeled nucleic acid probe to the cellular RNA in individual cells ortissues. Therefore, RNA in situ hybridization is a powerful approach forstudying tissue- and temporal-specific gene expression. In this method,oligonucleotides, cloned DNA fragments, or antisense RNA transcripts ofcloned DNA fragments corresponding to unique portions of heart of glassgenes are used to detect specific mRNA species, e.g., in the tissues ofanimals, such as mice, at various developmental stages. Other geneexpression detection techniques are known to those of skill in the artand can be employed for detection of heart of glass gene expression.

[0110] Identification of Additional Heart of Glass Genes

[0111] Standard techniques, such as the polymerase chain reaction (PCR)and DNA hybridization, can be used to clone heart of glass homologues inother species and heart of glass-related genes in humans. Heart ofglass-related genes and homologues can be readily identified usinglow-stringency DNA hybridization or low-stringency PCR with human heartof glass probes or primers. Degenerate primers encoding human heart ofglass or human heart of glass-related amino acid sequences can be usedto clone additional heart of glass-related genes and homologues byRT-PCR.

[0112] Construction of Transgenic Animals and Knockout Animals

[0113] Characterization of heart of glass genes provides informationthat allows heart of glass knockout animal models to be developed byhomologous recombination. Preferably, a heart of glass knockout animalis a mammal, most preferably a mouse. Similarly, animal models of heartof glass overproduction can be generated by integrating one or moreheart of glass sequences into the genome of an animal, according tostandard transgenic techniques. Moreover, the effect of heart of glassmutations (e.g., dominant gene mutations) can be studied usingtransgenic mice carrying mutated heart of glass transgenes or byintroducing such mutations into the endogenous heart of glass gene,using standard homologous recombination techniques.

[0114] A replacement-type targeting vector, which can be used to createa knockout model, can be constructed using an isogenic genomic clone,for example, from a mouse strain such as 129/Sv (Stratagene Inc.,LaJolla, Calif.). The targeting vector can be introduced into a suitablyderived line of embryonic stem (ES) cells by electroporation to generateES cell lines that carry a profoundly truncated form of heart of glassgene. To generate chimeric founder mice, the targeted cell lines areinjected into a mouse blastula-stage embryo. Heterozygous offspring canbe interbred to homozygosity. Heart of glass knockout mice provide atool for studying the role of heart of glass in embryonic developmentand in disease. Moreover, such mice provide the means, in vivo, fortesting therapeutic compounds for amelioration of diseases or conditionsinvolving heart of glass-dependent or a heart of glass-affected pathway.

[0115] Experimental Results

[0116] To better understand the basis of the heart of glass (heg)mutation, we initiated positional cloning projects and have determinedthe gene responsible for the heg mutation (FIGS. 1 and 2). This gene isnot dominated by any known structural motifs, but does possess two shortpeptide stretches that have the distinctive six cysteines associatedwith EGF-repeats (amino acids 580 to 660), which are found in manydifferent proteins of diverse function. As shown in FIG. 3, the proteinexhibits about 34% identity over the deduced 650 amino acids of the ESTKIAA1237 (Genbank entry BAA86551.1), and about 80% identity over theC-terminal 100 amino acids.

[0117] The mutation in the heart of glass gene is a stop codon resultingfrom a G to A change at residue 497, switching a tryptophan codon to astop codon (TGG to TAG). This mutation occurs at amino acid 103, earlyin the sequence, and would result in a dramatic protein truncation. Acleavable signal peptide is predicted based on the primary structure,with a possible cleavage site between amino acids 23 and 24. Thisstructure would still be encoded by the truncated protein and possiblycleaved, but the resulting peptide of 80 amino acids may not have anyphysiological function.

[0118] To characterize this gene, we have examined the expressionpattern in zebrafish embryos using whole mount in situ hybridization atseveral stages of development with the 3′ cDNA fragment isolated from a24 hour cDNA library. The gene is expressed in the heart at 24 hours and48 hours of development, but at other stages demonstrates a lessrestricted expression pattern. Further experiments indicate that heg isexpressed in the outflow tract of the heart and ventricle, in theendocardium.

[0119] Additional experiments include injection experiments usingmorphlino antisense oligonucleotides, to phenocopy the mutation toprovide further evidence that we have identified the mutated gene in hegembryos. Initial experiments reveal a loss of circulation in treatedembryos, as well as substantial pericardial edema. In several embryosthere is no hypertrophy, but there is edema and lack of circulation. Theantisense oligonucleotide may not completely block translation of theheg message, while the stop codon in the heg mutants may completelyprevent any functional heg protein from being synthesized. Thus, thephenotype associated with the morpholino injections may in fact be ahypermorphic response. Also, the phenotype is most obvious at 48 hoursof development, by which time the oligonucleotide may be losingactivity.

[0120] Other Embodiments

[0121] All publications and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

[0122] While the invention has been described in connection withspecific embodiments thereof, it is to be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and can be applied to theessential features hereinbefore set forth, and follows in the scope ofthe appended claims.

1 6 1 4519 DNA Danio rerio CDS (191)...(2713) 1 agcagtggta acaacgcagagtacgcgggg ctcgccgcct gaaataaaac ccttccaaga 60 cttcttcacc aactttaaatccatcccaag gaatttacta atgggaaact gttaaaagag 120 acccactttc cgatggtggcccggatcagt ttggcgtttt atttattagg aagcccgccc 180 ggtcgccttt atg atg gaaacg tgc gct cgc cgt gtg ctt ttc acg gcc 229 Met Met Glu Thr Cys Ala ArgArg Val Leu Phe Thr Ala 1 5 10 gct ctg ctc gtc ctc agt act gtg ata gcggaa act ttc tcc aca gac 277 Ala Leu Leu Val Leu Ser Thr Val Ile Ala GluThr Phe Ser Thr Asp 15 20 25 tcg gac acc gat aat cca ctg agt aca gaa actttt tat agc cga gca 325 Ser Asp Thr Asp Asn Pro Leu Ser Thr Glu Thr PheTyr Ser Arg Ala 30 35 40 45 agt ggc ttg aaa caa acc tcc tcc tgg cca gggaga gag gcc act gcc 373 Ser Gly Leu Lys Gln Thr Ser Ser Trp Pro Gly ArgGlu Ala Thr Ala 50 55 60 aca gcc gtg gat cta tcg agc gga ctg gga gag atgacg gag att ccc 421 Thr Ala Val Asp Leu Ser Ser Gly Leu Gly Glu Met ThrGlu Ile Pro 65 70 75 gcc agt gta tcc atc act gca gcc cga gag gga cat tcacca aaa cct 469 Ala Ser Val Ser Ile Thr Ala Ala Arg Glu Gly His Ser ProLys Pro 80 85 90 tta caa acc tct aca aat gca gcg gat tgg aag acc agt acgacc tca 517 Leu Gln Thr Ser Thr Asn Ala Ala Asp Trp Lys Thr Ser Thr ThrSer 95 100 105 gat gag aca aca gaa cat ctt caa tct gac act gag ctt acccat aat 565 Asp Glu Thr Thr Glu His Leu Gln Ser Asp Thr Glu Leu Thr HisAsn 110 115 120 125 gcg acc gcc caa tgg gag agt cca tca tca gca tct cacagc att acc 613 Ala Thr Ala Gln Trp Glu Ser Pro Ser Ser Ala Ser His SerIle Thr 130 135 140 agc cac cat cca gta aca gag aca cga acc gtg cga gatgta aca gat 661 Ser His His Pro Val Thr Glu Thr Arg Thr Val Arg Asp ValThr Asp 145 150 155 ctg ata gac atg gac acc aca gac tca gtc tcc cac actgat agc acc 709 Leu Ile Asp Met Asp Thr Thr Asp Ser Val Ser His Thr AspSer Thr 160 165 170 tac att tcc aca acc aac cga gtt gga gaa cgc aca ctgctc tca gtg 757 Tyr Ile Ser Thr Thr Asn Arg Val Gly Glu Arg Thr Leu LeuSer Val 175 180 185 atc tcc aac agc acc ttt gcg tac acc cag aac tca agcatc tct gat 805 Ile Ser Asn Ser Thr Phe Ala Tyr Thr Gln Asn Ser Ser IleSer Asp 190 195 200 205 gca gag tct caa acg tcc cca tgg gag gag aag acatca gga gcc acc 853 Ala Glu Ser Gln Thr Ser Pro Trp Glu Glu Lys Thr SerGly Ala Thr 210 215 220 caa gtc aat gag gaa act gaa gaa act gtg tca acagtg tcc gaa cag 901 Gln Val Asn Glu Glu Thr Glu Glu Thr Val Ser Thr ValSer Glu Gln 225 230 235 act gat ccc act ttt gaa ggc cgc aat acc acc agtgca act ctg gag 949 Thr Asp Pro Thr Phe Glu Gly Arg Asn Thr Thr Ser AlaThr Leu Glu 240 245 250 act gag cgg tca acg tta tcc caa ggc act gag tcacag aca gga caa 997 Thr Glu Arg Ser Thr Leu Ser Gln Gly Thr Glu Ser GlnThr Gly Gln 255 260 265 cct agt gtc aca gga cag acc gct aag gag gtg actgat atc gac aat 1045 Pro Ser Val Thr Gly Gln Thr Ala Lys Glu Val Thr AspIle Asp Asn 270 275 280 285 cca aat tca aca ccg cct ctt aca gtg acc agtagg gat gtt gag gaa 1093 Pro Asn Ser Thr Pro Pro Leu Thr Val Thr Ser ArgAsp Val Glu Glu 290 295 300 aca gat gcc aca tca gtg agc agt gag aca tcttat aca caa acc agc 1141 Thr Asp Ala Thr Ser Val Ser Ser Glu Thr Ser TyrThr Gln Thr Ser 305 310 315 agt gac tct gcc tcc tca atc ttg cct ttt acttct agc gaa cac aat 1189 Ser Asp Ser Ala Ser Ser Ile Leu Pro Phe Thr SerSer Glu His Asn 320 325 330 gtc act agc aca tcc caa gag agc cat aat tccaca ctc atc tat tcc 1237 Val Thr Ser Thr Ser Gln Glu Ser His Asn Ser ThrLeu Ile Tyr Ser 335 340 345 aca aat act ggt ggt tcc act gag ttt tcg actgga tca gta agc tct 1285 Thr Asn Thr Gly Gly Ser Thr Glu Phe Ser Thr GlySer Val Ser Ser 350 355 360 365 act gcc cat gaa gaa act gaa cgg tct tcaact cgc ata gta gat gag 1333 Thr Ala His Glu Glu Thr Glu Arg Ser Ser ThrArg Ile Val Asp Glu 370 375 380 aca acc ctt cat gat gtt act tct gca ccccca gtg ctt gaa gat gta 1381 Thr Thr Leu His Asp Val Thr Ser Ala Pro ProVal Leu Glu Asp Val 385 390 395 gcc aca act atc gat gac tcg ctt tcc aagttc cct tcc ggc caa tcg 1429 Ala Thr Thr Ile Asp Asp Ser Leu Ser Lys PhePro Ser Gly Gln Ser 400 405 410 cct acc atc cct aaa acc gat gac caa accaac aca caa gtg gtg cca 1477 Pro Thr Ile Pro Lys Thr Asp Asp Gln Thr AsnThr Gln Val Val Pro 415 420 425 aca tca act cat agg cca cag gtt aca gatgaa gcc acc gat gag gtg 1525 Thr Ser Thr His Arg Pro Gln Val Thr Asp GluAla Thr Asp Glu Val 430 435 440 445 tct aca gtt tac agt tct acc act acttta act acc aca act cct tct 1573 Ser Thr Val Tyr Ser Ser Thr Thr Thr LeuThr Thr Thr Thr Pro Ser 450 455 460 gtc acc act aga caa ctc caa cca cactac acc aca gtg caa acc caa 1621 Val Thr Thr Arg Gln Leu Gln Pro His TyrThr Thr Val Gln Thr Gln 465 470 475 aca caa cac act acc att gtt acc accgat atc att cag gta ctg cga 1669 Thr Gln His Thr Thr Ile Val Thr Thr AspIle Ile Gln Val Leu Arg 480 485 490 acg aca ccc tct aca gcc cat cat gtgcct aca ttg act acc agt gga 1717 Thr Thr Pro Ser Thr Ala His His Val ProThr Leu Thr Thr Ser Gly 495 500 505 cca cag gct cca agt aca gct gat tcttct gac gtc acc aca ttg cac 1765 Pro Gln Ala Pro Ser Thr Ala Asp Ser SerAsp Val Thr Thr Leu His 510 515 520 525 ttg gaa acc agc aca gcc acg ccgggg aac act acg gcg cac ggt gga 1813 Leu Glu Thr Ser Thr Ala Thr Pro GlyAsn Thr Thr Ala His Gly Gly 530 535 540 cgt gca aca acg cct ttt agc aagagc agc ccg ggt aga aca act gtg 1861 Arg Ala Thr Thr Pro Phe Ser Lys SerSer Pro Gly Arg Thr Thr Val 545 550 555 gta gtg acc act gga cat ctc actgac aaa agc act aca gag aca gga 1909 Val Val Thr Thr Gly His Leu Thr AspLys Ser Thr Thr Glu Thr Gly 560 565 570 agc gca acc acg cag atg cct cttaga aca tca gca tca cca ggt cat 1957 Ser Ala Thr Thr Gln Met Pro Leu ArgThr Ser Ala Ser Pro Gly His 575 580 585 gta tgt gga cct aaa acc tgt gcaaat gga ggt cat tgt gtt aga tca 2005 Val Cys Gly Pro Lys Thr Cys Ala AsnGly Gly His Cys Val Arg Ser 590 595 600 605 gct gaa gga agt tac tac tgtcag tgt ctc tcc gca tgg acc gga ccc 2053 Ala Glu Gly Ser Tyr Tyr Cys GlnCys Leu Ser Ala Trp Thr Gly Pro 610 615 620 ttc tgc act gaa gat gtg gacgag tgt gtg aac agt cca tgt cct cag 2101 Phe Cys Thr Glu Asp Val Asp GluCys Val Asn Ser Pro Cys Pro Gln 625 630 635 ggt tca gtg tgt gtc aac acaggt ggt tct ttc agc tgt gaa tgt gac 2149 Gly Ser Val Cys Val Asn Thr GlyGly Ser Phe Ser Cys Glu Cys Asp 640 645 650 ctg ggc ttt gac ctg gag gatggc cgc agt tgt aca caa gtc aag aca 2197 Leu Gly Phe Asp Leu Glu Asp GlyArg Ser Cys Thr Gln Val Lys Thr 655 660 665 ttt ttg ggc act ttc aca gtcaac aac tct ctt cat ctc aga aat tta 2245 Phe Leu Gly Thr Phe Thr Val AsnAsn Ser Leu His Leu Arg Asn Leu 670 675 680 685 ggt ctg cac gag cta cacaga gag atc caa cag ctg ctc aat gct tct 2293 Gly Leu His Glu Leu His ArgGlu Ile Gln Gln Leu Leu Asn Ala Ser 690 695 700 ctc tcc atc ttc cat ggttat aga cgc ttt acc ctg ggt aaa aga gat 2341 Leu Ser Ile Phe His Gly TyrArg Arg Phe Thr Leu Gly Lys Arg Asp 705 710 715 gga caa ggt gtg caa atccca gtg gtg agc atg ttt tca ctc tcc tcc 2389 Gly Gln Gly Val Gln Ile ProVal Val Ser Met Phe Ser Leu Ser Ser 720 725 730 aat gtg acc agc gcg gatgtt ttc aac agc atc cag atg tcc ctt aac 2437 Asn Val Thr Ser Ala Asp ValPhe Asn Ser Ile Gln Met Ser Leu Asn 735 740 745 aac tgc agc cgg aca tactcg cac tgt ccc att aaa ctt cag cac cag 2485 Asn Cys Ser Arg Thr Tyr SerHis Cys Pro Ile Lys Leu Gln His Gln 750 755 760 765 ctc tcc tat cac gtggag agc ctg tgc atg gcc cag aag acc aag tgt 2533 Leu Ser Tyr His Val GluSer Leu Cys Met Ala Gln Lys Thr Lys Cys 770 775 780 gat gtg cag tac tcagat tgc tcg gat att agc ggg att ccc aac tgt 2581 Asp Val Gln Tyr Ser AspCys Ser Asp Ile Ser Gly Ile Pro Asn Cys 785 790 795 cag tgc ctt cct gggtac ttt aaa agg aac cca gaa gac atg acc tgc 2629 Gln Cys Leu Pro Gly TyrPhe Lys Arg Asn Pro Glu Asp Met Thr Cys 800 805 810 aga gac tgt gga gatgga ctc aag ctt gtt aat ggc aaa tgt gtc gaa 2677 Arg Asp Cys Gly Asp GlyLeu Lys Leu Val Asn Gly Lys Cys Val Glu 815 820 825 tgc atg ttt gga tttgga ggt ttc aac tgc aat aat taaaagacaa 2723 Cys Met Phe Gly Phe Gly GlyPhe Asn Cys Asn Asn 830 835 840 aaacgacatc aacaagatca tcttcaaaagtggagagctt cagatgtcgc catacgcaga 2783 gtttcctaag agcaatcgcg tgtccatggagtggggtaga gagactatcg agatgcagga 2843 gaacggcagt acaaaaaacc tcctgcaaatgactgacatt tattactcgc ctgcactgag 2903 aaactccgac ctggagcgta atggtctgtatccattctcg ggccttcctg gttcaaggca 2963 ttcctgcatc tatccggctc agtggaacccttccttctta agcgacgatt cacgccgaag 3023 agactacttt tgacagcttt acccagcaggcatgcaatct tgatgctgca tgtaagctct 3083 cttggcctgt tgcagatcct gtagctcgattctgaccact aaaggccttt gaatgtgaaa 3143 atgagctggc aggaaaaggc cattatctgtgagactggat gaaagccgca gagccctggc 3203 ggggtcaatt tagcattcgg acaaagtgccacaaaaagat tttagtatgg agctgatgaa 3263 aggatgttct tcgctcccca cagaccgcttattattaagc gcaagtcagc gaatttagac 3323 tagccggagg aagaatacaa ccacaagatgcctgagctac aatctaaaat ttatattatg 3383 aaagtttgca ctatgaactt ctcacacagatattatttca aatggagaaa gccataaatg 3443 tatcatattt atacttggaa tgatttacccagctggtttg tactgtgtta aggttttgta 3503 aaggacaaaa tatctggatg tttgtttgagtatgtgcatc aatagctgaa gagataattt 3563 acccctagat tttaattctg tcattatttactgtcgtaca cggatgtcca aactcaatcc 3623 tggagtgctg gtgtcctgca ggctttagcttcaaccgtaa ttagacatct taatcagcta 3683 accaagctct atctaggcca ggggtgctcaaccctgtacc tggagatagg aatggctgac 3743 atgaaactga cgtttcaaca ccgtgtccagatcccgaaac actgcactga agcatgatcc 3803 gaaacaccca ggtctcactg gaagtactaggtcatccgga gaagtacttc tcgcatactg 3863 attttcgaat tctttgaatt cggacatactactcagctca catactgatt tttgcgtatt 3923 gcatagtatg gaagtatgca gttttggacgcagccacgat ctctgcaagt tagtcaggaa 3983 ctctcaccac ttcactaaat gacttgaagcttcaactcta caacgtgagg tttgatacct 4043 caatttggtt aactttttct tcgctgaagctattccagag aaatacataa aaatgaccac 4103 taggtgtcac tgtagagtgg ggtttctaaacgtttcgaag ctttgaaaca tttgctttga 4163 ctgtttcagt gtttcatgaa gcctcgctttgcccatcact acttggagat ctaccttctt 4223 acagagttca gctccaaccc tgatcaaacacacctgagcc aattaattag gacctgaaca 4283 gcactggata attacaggca ggtgactaggacacccctga tctaggcata ctagaaactt 4343 ccaggcaggt gtgttgaggc aagttagagctaaactatgc gggagtttgg gcacctttgc 4403 tctagtaatc ctttgttagg agttgtttgttacaaaatgt tcagctcttt gctgtaaaat 4463 ggtttaagaa tcaagttttt aaagcataaaaaaaaaaaaa aaaaaaaaaa aaaaaa 4519 2 841 PRT Danio rerio 2 Met Met GluThr Cys Ala Arg Arg Val Leu Phe Thr Ala Ala Leu Leu 1 5 10 15 Val LeuSer Thr Val Ile Ala Glu Thr Phe Ser Thr Asp Ser Asp Thr 20 25 30 Asp AsnPro Leu Ser Thr Glu Thr Phe Tyr Ser Arg Ala Ser Gly Leu 35 40 45 Lys GlnThr Ser Ser Trp Pro Gly Arg Glu Ala Thr Ala Thr Ala Val 50 55 60 Asp LeuSer Ser Gly Leu Gly Glu Met Thr Glu Ile Pro Ala Ser Val 65 70 75 80 SerIle Thr Ala Ala Arg Glu Gly His Ser Pro Lys Pro Leu Gln Thr 85 90 95 SerThr Asn Ala Ala Asp Trp Lys Thr Ser Thr Thr Ser Asp Glu Thr 100 105 110Thr Glu His Leu Gln Ser Asp Thr Glu Leu Thr His Asn Ala Thr Ala 115 120125 Gln Trp Glu Ser Pro Ser Ser Ala Ser His Ser Ile Thr Ser His His 130135 140 Pro Val Thr Glu Thr Arg Thr Val Arg Asp Val Thr Asp Leu Ile Asp145 150 155 160 Met Asp Thr Thr Asp Ser Val Ser His Thr Asp Ser Thr TyrIle Ser 165 170 175 Thr Thr Asn Arg Val Gly Glu Arg Thr Leu Leu Ser ValIle Ser Asn 180 185 190 Ser Thr Phe Ala Tyr Thr Gln Asn Ser Ser Ile SerAsp Ala Glu Ser 195 200 205 Gln Thr Ser Pro Trp Glu Glu Lys Thr Ser GlyAla Thr Gln Val Asn 210 215 220 Glu Glu Thr Glu Glu Thr Val Ser Thr ValSer Glu Gln Thr Asp Pro 225 230 235 240 Thr Phe Glu Gly Arg Asn Thr ThrSer Ala Thr Leu Glu Thr Glu Arg 245 250 255 Ser Thr Leu Ser Gln Gly ThrGlu Ser Gln Thr Gly Gln Pro Ser Val 260 265 270 Thr Gly Gln Thr Ala LysGlu Val Thr Asp Ile Asp Asn Pro Asn Ser 275 280 285 Thr Pro Pro Leu ThrVal Thr Ser Arg Asp Val Glu Glu Thr Asp Ala 290 295 300 Thr Ser Val SerSer Glu Thr Ser Tyr Thr Gln Thr Ser Ser Asp Ser 305 310 315 320 Ala SerSer Ile Leu Pro Phe Thr Ser Ser Glu His Asn Val Thr Ser 325 330 335 ThrSer Gln Glu Ser His Asn Ser Thr Leu Ile Tyr Ser Thr Asn Thr 340 345 350Gly Gly Ser Thr Glu Phe Ser Thr Gly Ser Val Ser Ser Thr Ala His 355 360365 Glu Glu Thr Glu Arg Ser Ser Thr Arg Ile Val Asp Glu Thr Thr Leu 370375 380 His Asp Val Thr Ser Ala Pro Pro Val Leu Glu Asp Val Ala Thr Thr385 390 395 400 Ile Asp Asp Ser Leu Ser Lys Phe Pro Ser Gly Gln Ser ProThr Ile 405 410 415 Pro Lys Thr Asp Asp Gln Thr Asn Thr Gln Val Val ProThr Ser Thr 420 425 430 His Arg Pro Gln Val Thr Asp Glu Ala Thr Asp GluVal Ser Thr Val 435 440 445 Tyr Ser Ser Thr Thr Thr Leu Thr Thr Thr ThrPro Ser Val Thr Thr 450 455 460 Arg Gln Leu Gln Pro His Tyr Thr Thr ValGln Thr Gln Thr Gln His 465 470 475 480 Thr Thr Ile Val Thr Thr Asp IleIle Gln Val Leu Arg Thr Thr Pro 485 490 495 Ser Thr Ala His His Val ProThr Leu Thr Thr Ser Gly Pro Gln Ala 500 505 510 Pro Ser Thr Ala Asp SerSer Asp Val Thr Thr Leu His Leu Glu Thr 515 520 525 Ser Thr Ala Thr ProGly Asn Thr Thr Ala His Gly Gly Arg Ala Thr 530 535 540 Thr Pro Phe SerLys Ser Ser Pro Gly Arg Thr Thr Val Val Val Thr 545 550 555 560 Thr GlyHis Leu Thr Asp Lys Ser Thr Thr Glu Thr Gly Ser Ala Thr 565 570 575 ThrGln Met Pro Leu Arg Thr Ser Ala Ser Pro Gly His Val Cys Gly 580 585 590Pro Lys Thr Cys Ala Asn Gly Gly His Cys Val Arg Ser Ala Glu Gly 595 600605 Ser Tyr Tyr Cys Gln Cys Leu Ser Ala Trp Thr Gly Pro Phe Cys Thr 610615 620 Glu Asp Val Asp Glu Cys Val Asn Ser Pro Cys Pro Gln Gly Ser Val625 630 635 640 Cys Val Asn Thr Gly Gly Ser Phe Ser Cys Glu Cys Asp LeuGly Phe 645 650 655 Asp Leu Glu Asp Gly Arg Ser Cys Thr Gln Val Lys ThrPhe Leu Gly 660 665 670 Thr Phe Thr Val Asn Asn Ser Leu His Leu Arg AsnLeu Gly Leu His 675 680 685 Glu Leu His Arg Glu Ile Gln Gln Leu Leu AsnAla Ser Leu Ser Ile 690 695 700 Phe His Gly Tyr Arg Arg Phe Thr Leu GlyLys Arg Asp Gly Gln Gly 705 710 715 720 Val Gln Ile Pro Val Val Ser MetPhe Ser Leu Ser Ser Asn Val Thr 725 730 735 Ser Ala Asp Val Phe Asn SerIle Gln Met Ser Leu Asn Asn Cys Ser 740 745 750 Arg Thr Tyr Ser His CysPro Ile Lys Leu Gln His Gln Leu Ser Tyr 755 760 765 His Val Glu Ser LeuCys Met Ala Gln Lys Thr Lys Cys Asp Val Gln 770 775 780 Tyr Ser Asp CysSer Asp Ile Ser Gly Ile Pro Asn Cys Gln Cys Leu 785 790 795 800 Pro GlyTyr Phe Lys Arg Asn Pro Glu Asp Met Thr Cys Arg Asp Cys 805 810 815 GlyAsp Gly Leu Lys Leu Val Asn Gly Lys Cys Val Glu Cys Met Phe 820 825 830Gly Phe Gly Gly Phe Asn Cys Asn Asn 835 840 3 977 PRT Danio rerio 3 MetMet Glu Thr Cys Ala Arg Arg Val Leu Phe Thr Ala Ala Leu Leu 1 5 10 15Val Leu Ser Thr Val Ile Ala Glu Thr Phe Ser Thr Asp Ser Asp Thr 20 25 30Asp Asn Pro Leu Ser Thr Glu Thr Phe Tyr Ser Arg Ala Ser Gly Leu 35 40 45Lys Gln Thr Ser Ser Trp Pro Gly Arg Glu Ala Thr Ala Thr Ala Val 50 55 60Asp Leu Ser Ser Gly Leu Gly Glu Met Thr Glu Ile Pro Ala Ser Val 65 70 7580 Ser Ile Thr Ala Ala Arg Glu Gly His Ser Pro Lys Pro Leu Gln Thr 85 9095 Ser Thr Asn Ala Ala Asp Trp Lys Thr Ser Thr Thr Ser Asp Glu Thr 100105 110 Thr Glu His Leu Gln Ser Asp Thr Glu Leu Thr His Asn Ala Thr Ala115 120 125 Gln Trp Glu Ser Pro Ser Ser Ala Ser His Ser Ile Thr Ser HisHis 130 135 140 Pro Val Thr Glu Thr Arg Thr Val Arg Asp Val Thr Asp LeuIle Asp 145 150 155 160 Met Asp Thr Thr Asp Ser Val Ser His Thr Asp SerThr Tyr Ile Ser 165 170 175 Thr Thr Asn Arg Val Gly Glu Arg Thr Leu LeuSer Val Ile Ser Asn 180 185 190 Ser Thr Phe Ala Tyr Thr Gln Asn Ser SerIle Ser Asp Ala Glu Ser 195 200 205 Gln Thr Ser Pro Trp Glu Glu Lys ThrSer Gly Ala Thr Gln Val Asn 210 215 220 Glu Glu Thr Glu Glu Thr Val SerThr Val Ser Glu Gln Thr Asp Pro 225 230 235 240 Thr Phe Glu Gly Arg AsnThr Thr Ser Ala Thr Leu Glu Thr Glu Arg 245 250 255 Ser Thr Leu Ser GlnGly Thr Glu Ser Gln Thr Gly Gln Pro Ser Val 260 265 270 Thr Gly Gln ThrAla Lys Glu Val Thr Asp Ile Asp Asn Pro Asn Ser 275 280 285 Thr Pro ProLeu Thr Val Thr Ser Arg Asp Val Glu Glu Thr Asp Ala 290 295 300 Thr SerVal Ser Ser Glu Thr Ser Tyr Thr Gln Thr Ser Ser Asp Ser 305 310 315 320Ala Ser Ser Ile Leu Pro Phe Thr Ser Ser Glu His Asn Val Thr Ser 325 330335 Thr Ser Gln Glu Ser His Asn Ser Thr Leu Ile Tyr Ser Thr Asn Thr 340345 350 Gly Gly Ser Thr Glu Phe Ser Thr Gly Ser Val Ser Ser Thr Ala His355 360 365 Glu Glu Thr Glu Arg Ser Ser Thr Arg Ile Val Asp Glu Thr ThrLeu 370 375 380 His Asp Val Thr Ser Ala Pro Pro Val Leu Glu Asp Val AlaThr Thr 385 390 395 400 Ile Asp Asp Ser Leu Ser Lys Phe Pro Ser Gly GlnSer Pro Thr Ile 405 410 415 Pro Lys Thr Asp Asp Gln Thr Asn Thr Gln ValVal Pro Thr Ser Thr 420 425 430 His Arg Pro Gln Val Thr Asp Glu Ala ThrAsp Glu Val Ser Thr Val 435 440 445 Tyr Ser Ser Thr Thr Thr Leu Thr ThrThr Thr Pro Ser Val Thr Thr 450 455 460 Arg Gln Leu Gln Pro His Tyr ThrThr Val Gln Thr Gln Thr Gln His 465 470 475 480 Thr Thr Ile Val Thr ThrAsp Ile Ile Gln Val Leu Arg Thr Thr Pro 485 490 495 Ser Thr Ala His HisVal Pro Thr Leu Thr Thr Ser Gly Pro Gln Ala 500 505 510 Pro Ser Thr AlaAsp Ser Ser Asp Val Thr Thr Leu His Leu Glu Thr 515 520 525 Ser Thr AlaThr Pro Gly Asn Thr Thr Ala His Gly Gly Arg Ala Thr 530 535 540 Thr ProPhe Ser Lys Ser Ser Pro Gly Arg Thr Thr Val Val Val Thr 545 550 555 560Thr Gly His Leu Thr Asp Lys Ser Thr Thr Glu Thr Gly Ser Ala Thr 565 570575 Thr Gln Met Pro Leu Arg Thr Ser Ala Ser Pro Gly His Val Cys Gly 580585 590 Pro Lys Thr Cys Ala Asn Gly Gly His Cys Val Arg Ser Ala Glu Gly595 600 605 Ser Tyr Tyr Cys Gln Cys Leu Ser Ala Trp Thr Gly Pro Phe CysThr 610 615 620 Glu Asp Val Asp Glu Cys Val Asn Ser Pro Cys Pro Gln GlySer Val 625 630 635 640 Cys Val Asn Thr Gly Gly Ser Phe Ser Cys Glu CysAsp Leu Gly Phe 645 650 655 Asp Leu Glu Asp Gly Arg Ser Cys Thr Gln ValLys Thr Phe Leu Gly 660 665 670 Thr Phe Thr Val Asn Asn Ser Leu His LeuArg Asn Leu Gly Leu His 675 680 685 Glu Leu His Arg Glu Ile Gln Gln LeuLeu Asn Ala Ser Leu Ser Ile 690 695 700 Phe His Gly Tyr Arg Arg Phe ThrLeu Gly Lys Arg Asp Gly Gln Gly 705 710 715 720 Val Gln Ile Pro Val ValSer Met Phe Ser Leu Ser Ser Asn Val Thr 725 730 735 Ser Ala Asp Val PheAsn Ser Ile Gln Met Ser Leu Asn Asn Cys Ser 740 745 750 Arg Thr Tyr SerHis Cys Pro Ile Lys Leu Gln His Gln Leu Ser Tyr 755 760 765 His Val GluSer Leu Cys Met Ala Gln Lys Thr Lys Cys Asp Val Gln 770 775 780 Tyr SerAsp Cys Ser Asp Ile Ser Gly Val Pro Asn Cys Gln Cys Leu 785 790 795 800Pro Gly Tyr Phe Lys Arg Asn Pro Glu Asp Met Thr Cys Arg Asp Cys 805 810815 Gly Asp Gly Leu Lys Leu Val Asn Gly Lys Cys Val Glu Cys Met Phe 820825 830 Gly Phe Gly Gly Phe Asn Cys Asn Asn Phe Tyr Lys Leu Ile Ala Val835 840 845 Val Val Ser Pro Ala Gly Gly Ala Leu Leu Leu Ile Val Val IleAla 850 855 860 Leu Ile Val Thr Cys Cys Lys Lys Asp Lys Asn Asp Ile AsnLys Ile 865 870 875 880 Ile Phe Lys Ser Gly Glu Leu Gln Met Ser Pro TyrAla Glu Phe Pro 885 890 895 Lys Ser Asn Arg Val Ser Met Glu Trp Gly ArgGlu Thr Ile Glu Met 900 905 910 Gln Glu Asn Gly Ser Thr Lys Asn Leu LeuGln Met Thr Asp Ile Tyr 915 920 925 Tyr Ser Pro Ala Leu Arg Asn Ser AspLeu Glu Arg Asn Gly Leu Tyr 930 935 940 Pro Phe Ser Gly Leu Pro Gly SerArg His Ser Cys Ile Tyr Pro Ala 945 950 955 960 Gln Trp Asn Pro Ser PheLeu Ser Asp Asp Ser Arg Arg Arg Asp Tyr 965 970 975 Phe 4 4518 DNA Homosapiens CDS (2)...(1963) 4 a gca gtg gta aca acg cag agt acg cgg ggc tcgccg cct gaa ata aaa 49 Ala Val Val Thr Thr Gln Ser Thr Arg Gly Ser ProPro Glu Ile Lys 1 5 10 15 ccc ttc caa gac ttc ttc acc aac ttt aaa tccatc cca agg aat tta 97 Pro Phe Gln Asp Phe Phe Thr Asn Phe Lys Ser IlePro Arg Asn Leu 20 25 30 cta atg gga aac tgt taa aag aga ccc act ttc cgatgg tgg ccc gga 145 Leu Met Gly Asn Cys * Lys Arg Pro Thr Phe Arg TrpTrp Pro Gly 35 40 45 tca gtt tgg cgt ttt att tat tag gaa gcc cgc ccg gtcgcc ttt atg 193 Ser Val Trp Arg Phe Ile Tyr * Glu Ala Arg Pro Val AlaPhe Met 50 55 60 atg gaa acg tgc gct cgc cgt gtg ctt ttc acg gcc gct ctgctc gtc 241 Met Glu Thr Cys Ala Arg Arg Val Leu Phe Thr Ala Ala Leu LeuVal 65 70 75 ctc agt act gtg ata gcg gaa act ttc tcc aca gac tcg gac accgat 289 Leu Ser Thr Val Ile Ala Glu Thr Phe Ser Thr Asp Ser Asp Thr Asp80 85 90 aat cca ctg agt aca gaa act ttt tat agc cga gca agt ggc ttg aaa337 Asn Pro Leu Ser Thr Glu Thr Phe Tyr Ser Arg Ala Ser Gly Leu Lys 95100 105 110 caa acc tcc tcc tgg cca ggg aga gag gcc act gcc aca gcc gtggat 385 Gln Thr Ser Ser Trp Pro Gly Arg Glu Ala Thr Ala Thr Ala Val Asp115 120 125 cta tcg agc gga ctg gga gag atg acg gag att ccc gcc agt gtatcc 433 Leu Ser Ser Gly Leu Gly Glu Met Thr Glu Ile Pro Ala Ser Val Ser130 135 140 atc act gca gcc cga gag gga cat tca cca aaa cct tta caa acctct 481 Ile Thr Ala Ala Arg Glu Gly His Ser Pro Lys Pro Leu Gln Thr Ser145 150 155 aca aat gca gcg gat tgg aag acc agt acg acc tca gat gag acaaca 529 Thr Asn Ala Ala Asp Trp Lys Thr Ser Thr Thr Ser Asp Glu Thr Thr160 165 170 gaa cat ctt caa tct gac act gag ctt acc cat aat gcg acc gcccaa 577 Glu His Leu Gln Ser Asp Thr Glu Leu Thr His Asn Ala Thr Ala Gln175 180 185 190 tgg gag agt cca tca tca gca tct cac agc att acc agc caccat cca 625 Trp Glu Ser Pro Ser Ser Ala Ser His Ser Ile Thr Ser His HisPro 195 200 205 gta aca gag aca cga acc gtg cga gat gta aca gat ctg atagac atg 673 Val Thr Glu Thr Arg Thr Val Arg Asp Val Thr Asp Leu Ile AspMet 210 215 220 gac acc aca gac tca gtc tcc cac act gat agc acc tac atttcc aca 721 Asp Thr Thr Asp Ser Val Ser His Thr Asp Ser Thr Tyr Ile SerThr 225 230 235 acc aac cga gtt gga gaa cgc aca ctg ctc tca gtg atc tccaac agc 769 Thr Asn Arg Val Gly Glu Arg Thr Leu Leu Ser Val Ile Ser AsnSer 240 245 250 acc ttt gcg tac acc cag aac tca agc atc tct gat gca gagtct caa 817 Thr Phe Ala Tyr Thr Gln Asn Ser Ser Ile Ser Asp Ala Glu SerGln 255 260 265 270 acg tcc cca tgg gag gag aag aca tca gga gcc acc caagtc aat gag 865 Thr Ser Pro Trp Glu Glu Lys Thr Ser Gly Ala Thr Gln ValAsn Glu 275 280 285 gaa act gaa gaa act gtg tca aca gtg tcc gaa cag actgat ccc act 913 Glu Thr Glu Glu Thr Val Ser Thr Val Ser Glu Gln Thr AspPro Thr 290 295 300 ttt gaa ggc cgc aat acc acc agt gca act ctg gag actgag cgg tca 961 Phe Glu Gly Arg Asn Thr Thr Ser Ala Thr Leu Glu Thr GluArg Ser 305 310 315 acg tta tcc caa ggc act gag tca cag aca gga caa cctagt gtc aca 1009 Thr Leu Ser Gln Gly Thr Glu Ser Gln Thr Gly Gln Pro SerVal Thr 320 325 330 gga cag acc gct aag gag gtg act gat atc gac aat ccaaat tca aca 1057 Gly Gln Thr Ala Lys Glu Val Thr Asp Ile Asp Asn Pro AsnSer Thr 335 340 345 350 ccg cct ctt aca gtg acc agt agg gat gtt gag gaaaca gat gcc aca 1105 Pro Pro Leu Thr Val Thr Ser Arg Asp Val Glu Glu ThrAsp Ala Thr 355 360 365 tca gtg agc agt gag aca tct tat aca caa acc agcagt gac tct gcc 1153 Ser Val Ser Ser Glu Thr Ser Tyr Thr Gln Thr Ser SerAsp Ser Ala 370 375 380 tcc tca atc ttg cct ttt act tct agc gaa cac aatgtc act agc aca 1201 Ser Ser Ile Leu Pro Phe Thr Ser Ser Glu His Asn ValThr Ser Thr 385 390 395 tcc caa gag agc cat aat tcc aca ctc atc tat tccaca aat act ggt 1249 Ser Gln Glu Ser His Asn Ser Thr Leu Ile Tyr Ser ThrAsn Thr Gly 400 405 410 ggt tcc act gag ttt tcg act gga tca gta agc tctact gcc cat gaa 1297 Gly Ser Thr Glu Phe Ser Thr Gly Ser Val Ser Ser ThrAla His Glu 415 420 425 430 gaa act gaa cgg tct tca act cgc ata gta gatgag aca acc ctt cat 1345 Glu Thr Glu Arg Ser Ser Thr Arg Ile Val Asp GluThr Thr Leu His 435 440 445 gat gtt act tct gca ccc cca gtg ctt gaa gatgta gcc aca act atc 1393 Asp Val Thr Ser Ala Pro Pro Val Leu Glu Asp ValAla Thr Thr Ile 450 455 460 gat gac tcg ctt tcc aag ttc cct tcc ggc caatcg cct acc atc cct 1441 Asp Asp Ser Leu Ser Lys Phe Pro Ser Gly Gln SerPro Thr Ile Pro 465 470 475 aaa acc gat gac caa acc aac aca caa gtg gtgcca aca tca act cat 1489 Lys Thr Asp Asp Gln Thr Asn Thr Gln Val Val ProThr Ser Thr His 480 485 490 agg cca cag gtt aca gat gaa gcc acc gat gaggtg tct aca gtt tac 1537 Arg Pro Gln Val Thr Asp Glu Ala Thr Asp Glu ValSer Thr Val Tyr 495 500 505 510 agt tct acc act act tta act acc aca actcct tct gtc acc act aga 1585 Ser Ser Thr Thr Thr Leu Thr Thr Thr Thr ProSer Val Thr Thr Arg 515 520 525 caa ctc caa cca cac tac acc aca gtg caaacc caa aca caa cac act 1633 Gln Leu Gln Pro His Tyr Thr Thr Val Gln ThrGln Thr Gln His Thr 530 535 540 acc att gtt acc acc gat atc att cag gtactg cga acg aca ccc tct 1681 Thr Ile Val Thr Thr Asp Ile Ile Gln Val LeuArg Thr Thr Pro Ser 545 550 555 aca gcc cat cat gtg cct aca ttg act accagt gga cca cag gct cca 1729 Thr Ala His His Val Pro Thr Leu Thr Thr SerGly Pro Gln Ala Pro 560 565 570 agt aca gct gat tct tct gac gtc acc acattg cac ttg gaa acc agc 1777 Ser Thr Ala Asp Ser Ser Asp Val Thr Thr LeuHis Leu Glu Thr Ser 575 580 585 590 aca gcc acg ccg ggg aac act acg gcgcac ggt gga cgt gca aca acg 1825 Thr Ala Thr Pro Gly Asn Thr Thr Ala HisGly Gly Arg Ala Thr Thr 595 600 605 cct ttt agc aag agc agc ccg ggt agaaca act gtg gta gtg acc act 1873 Pro Phe Ser Lys Ser Ser Pro Gly Arg ThrThr Val Val Val Thr Thr 610 615 620 gga cat ctc act gac aaa agc act acagag aca gga agc gca acc acg 1921 Gly His Leu Thr Asp Lys Ser Thr Thr GluThr Gly Ser Ala Thr Thr 625 630 635 cag atg cct ctt aga aca tca gca tcacca ggt cat gta tgt 1963 Gln Met Pro Leu Arg Thr Ser Ala Ser Pro Gly HisVal Cys 640 645 650 ggacctaaaa cctgtgcaaa tggaggtcat tgtgttagatcagctgaagg aagttactac 2023 tgtcagtgtc tctccgcatg gaccggaccc ttctgcactgaagatgtgga cgagtgtgtg 2083 aacagtccat gtcctcaggg ttcagtgtgt gtcaacacaggtggttcttt cagctgtgaa 2143 tgtgacctgg gctttgacct ggaggatggc cgcagttgtacacaagtcaa gacatttttg 2203 ggcactttca cagtcaacaa ctctcttcat ctcagaaatttaggtctgca cgagctacac 2263 agagagatcc aacagctgct caatgcttct ctctccatcttccatggtta tagacgcttt 2323 accctgggta aaagagatgg acaaggtgtg caaatcccagtggtgagcat gttttcactc 2383 tcctccaatg tgaccagcgc ggatgttttc aacagcatccagatgtccct taacaactgc 2443 agccggacat actcgcactg tcccattaaa cttcagcaccagctctccta tcacgtggag 2503 agcctgtgca tggcccagaa gaccaagtgt gatgtgcagtactcagattg ctcggatatt 2563 agcgggattc ccaactgtca gtgccttcct gggtactttaaaaggaaccc agaagacatg 2623 acctgcagag actgtggaga tggactcaag cttgttaatggcaaatgtgt cgaatgcatg 2683 tttggatttg gaggtttcaa ctgcaataat taaagacaaaaacgacatca acaagatcat 2743 cttcaaaagt ggagagcttc agatgtcgcc atacgcagagtttcctaaga gcaatcgcgt 2803 gtccatggag tggggtagag agactatcga gatgcaggagaacggcagta caaaaaacct 2863 cctgcaaatg actgacattt attactcgcc tgcactgagaaactccgacc tggagcgtaa 2923 tggtctgtat ccattctcgg gccttcctgg ttcaaggcattcctgcatct atccggctca 2983 gtggaaccct tccttcttaa gcgacgattc acgccgaagagactactttt gacagcttta 3043 cccagcaggc atgcaatctt gatgctgcat gtaagctctcttggcctgtt gcagatcctg 3103 tagctcgatt ctgaccacta aaggcctttg aatgtgaaaatgagctggca ggaaaaggcc 3163 attatctgtg agactggatg aaagccgcag agccctggcggggtcaattt agcattcgga 3223 caaagtgcca caaaaagatt ttagtatgga gctgatgaaaggatgttctt cgctccccac 3283 agaccgctta ttattaagcg caagtcagcg aatttagactagccggagga agaatacaac 3343 cacaagatgc ctgagctaca atctaaaatt tatattatgaaagtttgcac tatgaacttc 3403 tcacacagat attatttcaa atggagaaag ccataaatgtatcatattta tacttggaat 3463 gatttaccca gctggtttgt actgtgttaa ggttttgtaaaggacaaaat atctggatgt 3523 ttgtttgagt atgtgcatca atagctgaag agataatttacccctagatt ttaattctgt 3583 cattatttac tgtcgtacac ggatgtccaa actcaatcctggagtgctgg tgtcctgcag 3643 gctttagctt caaccgtaat tagacatctt aatcagctaaccaagctcta tctaggccag 3703 gggtgctcaa ccctgtacct ggagatagga atggctgacatgaaactgac gtttcaacac 3763 cgtgtccaga tcccgaaaca ctgcactgaa gcatgatccgaaacacccag gtctcactgg 3823 aagtactagg tcatccggag aagtacttct cgcatactgattttcgaatt ctttgaattc 3883 ggacatacta ctcagctcac atactgattt ttgcgtattgcatagtatgg aagtatgcag 3943 ttttggacgc agccacgatc tctgcaagtt agtcaggaactctcaccact tcactaaatg 4003 acttgaagct tcaactctac aacgtgaggt ttgatacctcaatttggtta actttttctt 4063 cgctgaagct attccagaga aatacataaa aatgaccactaggtgtcact gtagagtggg 4123 gtttctaaac gtttcgaagc tttgaaacat ttgctttgactgtttcagtg tttcatgaag 4183 cctcgctttg cccatcacta cttggagatc taccttcttacagagttcag ctccaaccct 4243 gatcaaacac acctgagcca attaattagg acctgaacagcactggataa ttacaggcag 4303 gtgactagga cacccctgat ctaggcatac tagaaacttccaggcaggtg tgttgaggca 4363 agttagagct aaactatgcg ggagtttggg cacctttgctctagtaatcc tttgttagga 4423 gttgtttgtt acaaaatgtt cagctctttg ctgtaaaatggtttaagaat caagttttta 4483 aagcataaaa aaaaaaaaaa aaaaaaaaaa aaaaa 4518 5652 PRT Homo sapiens 5 Ala Val Val Thr Thr Gln Ser Thr Arg Gly Ser ProPro Glu Ile Lys 1 5 10 15 Pro Phe Gln Asp Phe Phe Thr Asn Phe Lys SerIle Pro Arg Asn Leu 20 25 30 Leu Met Gly Asn Cys Lys Arg Pro Thr Phe ArgTrp Trp Pro Gly Ser 35 40 45 Val Trp Arg Phe Ile Tyr Glu Ala Arg Pro ValAla Phe Met Met Glu 50 55 60 Thr Cys Ala Arg Arg Val Leu Phe Thr Ala AlaLeu Leu Val Leu Ser 65 70 75 80 Thr Val Ile Ala Glu Thr Phe Ser Thr AspSer Asp Thr Asp Asn Pro 85 90 95 Leu Ser Thr Glu Thr Phe Tyr Ser Arg AlaSer Gly Leu Lys Gln Thr 100 105 110 Ser Ser Trp Pro Gly Arg Glu Ala ThrAla Thr Ala Val Asp Leu Ser 115 120 125 Ser Gly Leu Gly Glu Met Thr GluIle Pro Ala Ser Val Ser Ile Thr 130 135 140 Ala Ala Arg Glu Gly His SerPro Lys Pro Leu Gln Thr Ser Thr Asn 145 150 155 160 Ala Ala Asp Trp LysThr Ser Thr Thr Ser Asp Glu Thr Thr Glu His 165 170 175 Leu Gln Ser AspThr Glu Leu Thr His Asn Ala Thr Ala Gln Trp Glu 180 185 190 Ser Pro SerSer Ala Ser His Ser Ile Thr Ser His His Pro Val Thr 195 200 205 Glu ThrArg Thr Val Arg Asp Val Thr Asp Leu Ile Asp Met Asp Thr 210 215 220 ThrAsp Ser Val Ser His Thr Asp Ser Thr Tyr Ile Ser Thr Thr Asn 225 230 235240 Arg Val Gly Glu Arg Thr Leu Leu Ser Val Ile Ser Asn Ser Thr Phe 245250 255 Ala Tyr Thr Gln Asn Ser Ser Ile Ser Asp Ala Glu Ser Gln Thr Ser260 265 270 Pro Trp Glu Glu Lys Thr Ser Gly Ala Thr Gln Val Asn Glu GluThr 275 280 285 Glu Glu Thr Val Ser Thr Val Ser Glu Gln Thr Asp Pro ThrPhe Glu 290 295 300 Gly Arg Asn Thr Thr Ser Ala Thr Leu Glu Thr Glu ArgSer Thr Leu 305 310 315 320 Ser Gln Gly Thr Glu Ser Gln Thr Gly Gln ProSer Val Thr Gly Gln 325 330 335 Thr Ala Lys Glu Val Thr Asp Ile Asp AsnPro Asn Ser Thr Pro Pro 340 345 350 Leu Thr Val Thr Ser Arg Asp Val GluGlu Thr Asp Ala Thr Ser Val 355 360 365 Ser Ser Glu Thr Ser Tyr Thr GlnThr Ser Ser Asp Ser Ala Ser Ser 370 375 380 Ile Leu Pro Phe Thr Ser SerGlu His Asn Val Thr Ser Thr Ser Gln 385 390 395 400 Glu Ser His Asn SerThr Leu Ile Tyr Ser Thr Asn Thr Gly Gly Ser 405 410 415 Thr Glu Phe SerThr Gly Ser Val Ser Ser Thr Ala His Glu Glu Thr 420 425 430 Glu Arg SerSer Thr Arg Ile Val Asp Glu Thr Thr Leu His Asp Val 435 440 445 Thr SerAla Pro Pro Val Leu Glu Asp Val Ala Thr Thr Ile Asp Asp 450 455 460 SerLeu Ser Lys Phe Pro Ser Gly Gln Ser Pro Thr Ile Pro Lys Thr 465 470 475480 Asp Asp Gln Thr Asn Thr Gln Val Val Pro Thr Ser Thr His Arg Pro 485490 495 Gln Val Thr Asp Glu Ala Thr Asp Glu Val Ser Thr Val Tyr Ser Ser500 505 510 Thr Thr Thr Leu Thr Thr Thr Thr Pro Ser Val Thr Thr Arg GlnLeu 515 520 525 Gln Pro His Tyr Thr Thr Val Gln Thr Gln Thr Gln His ThrThr Ile 530 535 540 Val Thr Thr Asp Ile Ile Gln Val Leu Arg Thr Thr ProSer Thr Ala 545 550 555 560 His His Val Pro Thr Leu Thr Thr Ser Gly ProGln Ala Pro Ser Thr 565 570 575 Ala Asp Ser Ser Asp Val Thr Thr Leu HisLeu Glu Thr Ser Thr Ala 580 585 590 Thr Pro Gly Asn Thr Thr Ala His GlyGly Arg Ala Thr Thr Pro Phe 595 600 605 Ser Lys Ser Ser Pro Gly Arg ThrThr Val Val Val Thr Thr Gly His 610 615 620 Leu Thr Asp Lys Ser Thr ThrGlu Thr Gly Ser Ala Thr Thr Gln Met 625 630 635 640 Pro Leu Arg Thr SerAla Ser Pro Gly His Val Cys 645 650 6 654 PRT Homo sapiens 6 Ser Ala ProLeu Ser Val Ser Gln Thr Thr Leu Pro Gln Ser Ser Ser 1 5 10 15 Thr ProVal Leu Pro Arg Ala Arg Glu Thr Pro Val Thr Ser Phe Gln 20 25 30 Thr SerThr Met Thr Ser Phe Met Thr Met Leu His Ser Ser Gln Thr 35 40 45 Ala AspLeu Lys Ser Gln Ser Thr Pro His Gln Glu Lys Val Ile Thr 50 55 60 Glu SerLys Ser Pro Ser Leu Val Ser Leu Pro Thr Glu Ser Thr Lys 65 70 75 80 AlaVal Thr Thr Asn Ser Pro Leu Pro Pro Ser Leu Thr Glu Ser Ser 85 90 95 ThrGlu Gln Thr Leu Pro Ala Thr Ser Thr Asn Leu Ala Gln Met Ser 100 105 110Pro Thr Phe Thr Thr Thr Ile Leu Lys Thr Ser Gln Pro Leu Met Thr 115 120125 Thr Pro Gly Thr Leu Ser Ser Thr Ala Ser Leu Val Thr Gly Pro Ile 130135 140 Ala Val Gln Thr Thr Ala Gly Lys Gln Leu Ser Leu Thr His Pro Glu145 150 155 160 Ile Leu Val Pro Gln Ile Ser Thr Glu Gly Gly Ile Ser ThrGlu Arg 165 170 175 Asn Arg Val Ile Val Asp Ala Thr Thr Gly Leu Ile ProLeu Thr Ser 180 185 190 Val Pro Thr Ser Ala Lys Glu Met Thr Thr Lys LeuGly Val Thr Ala 195 200 205 Glu Tyr Ser Pro Ala Ser Arg Ser Leu Gly ThrSer Pro Ser Pro Gln 210 215 220 Thr Thr Val Val Ser Thr Ala Glu Asp LeuAla Pro Lys Ser Ala Thr 225 230 235 240 Phe Ala Val Gln Ser Ser Thr GlnSer Pro Thr Thr Leu Ser Ser Ser 245 250 255 Ala Ser Val Asn Ser Cys AlaVal Asn Pro Cys Leu His Asn Gly Glu 260 265 270 Cys Val Ala Asp Asn ThrSer Arg Gly Tyr His Cys Arg Cys Pro Pro 275 280 285 Ser Trp Gln Gly AspAsp Cys Ser Val Asp Val Asn Glu Cys Leu Ser 290 295 300 Asn Pro Cys ProSer Thr Ala Thr Cys Asn Asn Thr Gln Gly Ser Phe 305 310 315 320 Ile CysLys Cys Pro Val Gly Tyr Gln Leu Glu Lys Gly Ile Cys Asn 325 330 335 LeuVal Arg Thr Phe Val Thr Glu Phe Lys Leu Lys Arg Thr Phe Leu 340 345 350Asn Thr Thr Val Glu Lys His Ser Asp Leu Gln Glu Val Glu Asn Glu 355 360365 Ile Thr Lys Thr Leu Asn Met Cys Phe Ser Ala Leu Pro Ser Tyr Ile 370375 380 Arg Ser Thr Val His Ala Ser Arg Glu Ser Asn Ala Val Val Ile Ser385 390 395 400 Leu Gln Thr Thr Phe Ser Leu Ala Ser Asn Val Thr Leu PheAsp Leu 405 410 415 Ala Asp Arg Met Gln Lys Cys Val Asn Ser Cys Lys SerSer Ala Glu 420 425 430 Val Cys Gln Leu Leu Gly Ser Gln Arg Arg Ile PheArg Ala Gly Ser 435 440 445 Leu Cys Lys Arg Lys Ser Pro Glu Cys Asp LysAsp Thr Ser Ile Cys 450 455 460 Thr Asp Leu Asp Gly Val Ala Leu Cys GlnCys Lys Ser Gly Tyr Phe 465 470 475 480 Gln Phe Asn Lys Met Asp His SerCys Arg Ala Cys Glu Asp Gly Tyr 485 490 495 Arg Leu Glu Asn Glu Thr CysMet Ser Cys Pro Phe Gly Leu Gly Gly 500 505 510 Leu Asn Cys Gly Asn ProTyr Gln Leu Ile Thr Val Val Ile Ala Ala 515 520 525 Ala Gly Gly Gly LeuLeu Leu Ile Leu Gly Ile Ala Leu Ile Val Thr 530 535 540 Cys Cys Arg LysAsn Lys Asn Asp Ile Ser Lys Leu Ile Phe Lys Ser 545 550 555 560 Gly AspPhe Gln Met Ser Pro Tyr Ala Glu Tyr Pro Lys Asn Pro Arg 565 570 575 SerGln Glu Trp Gly Arg Glu Ala Ile Glu Met His Glu Asn Gly Ser 580 585 590Thr Lys Asn Leu Leu Gln Met Thr Asp Val Tyr Tyr Ser Pro Thr Ser 595 600605 Val Arg Asn Pro Glu Leu Glu Arg Asn Gly Leu Tyr Pro Ala Tyr Thr 610615 620 Gly Leu Pro Gly Ser Arg His Ser Cys Ile Phe Pro Gly Gln Tyr Asn625 630 635 640 Pro Ser Phe Ile Ser Asp Glu Ser Arg Arg Arg Asp Tyr Phe645 650

What is claimed is:
 1. A method of determining whether a test subjecthas, or is at risk of developing, a disease or condition related to aheart of glass protein, said method comprising analyzing a nucleic acidmolecule of a sample from the test subject to determine whether the testsubject has a mutation in a gene encoding said protein, wherein thepresence of a mutation indicates that said test subject has, or is atrisk of developing, a disease related to a heart of glass protein. 2.The method of claim 1, further comprising the step of using nucleic acidmolecule primers specific for a gene encoding a heart of glass proteinfor nucleic acid molecule amplification of the gene by the polymerasechain reaction.
 3. The method of claim 1, wherein determination ofwhether said gene comprises a mutation is carried out by sequencing anucleic acid molecule encoding a heart of glass protein from said testsubject.
 4. The method of claim 1, wherein said test subject is amammal.
 5. The method of claim 1, wherein said test subject is a human.6. The method of claim 1, wherein said disease or condition is heartdisease.
 7. The method of claim 1, wherein said disease or condition isheart failure.
 8. The method of claim 1, wherein said mutation is theheart of glass mutation.
 9. A method for identifying a compound that canbe used to treat or to prevent heart disease, said method comprisingcontacting an organism comprising a mutation in a gene encoding a heartof glass protein and having a phenotype characteristic of heart diseasewith said compound, and determining the effect of said compound on saidphenotype, wherein detection of an improvement in said phenotypeindicates the identification of a compound that can be used to treat orto prevent heart disease.
 10. The method of claim 9, wherein said heartdisease is heart failure.
 11. The method of claim 9, wherein saidorganism is a zebrafish.
 12. The method of claim 9, wherein saidmutation in the gene encoding the heart of glass protein is the heart ofglass mutation.
 13. A method of treating or preventing heart disease ina patient, said method comprising administering to said patient acompound identified using the method of claim
 9. 14. The method of claim13, wherein said heart disease is heart failure.
 15. The method of claim13, wherein said patient has a mutation in a gene encoding a heart ofglass protein.
 16. The method of claim 15, wherein said mutation is theheart of glass mutation.
 17. A method of treating or preventing heartdisease in a patient, said method comprising administering to saidpatient a functional heart of glass protein or an expression vectorcomprising a nucleic acid molecule encoding said protein.
 18. Asubstantially pure zebrafish heart of glass polypeptide.
 19. Thepolypeptide of claim 18, wherein said polypeptide comprises an aminoacid sequence that is substantially identical to the amino acid sequenceof SEQ ID NO:2 or SEQ ID NO:3.
 20. The polypeptide of claim 19, whereinsaid polypeptide comprises the amino acid sequence of SEQ ID NO:2 or SEQID NO:3.
 21. A substantially pure nucleic acid molecule comprising asequence encoding a zebrafish heart of glass polypeptide.
 22. Thenucleic acid molecule of claim 21, wherein said nucleic acid moleculeencodes a polypeptide comprising an amino sequence that is substantiallyidentical to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3. 23.The nucleic acid molecule of claim 21, wherein said nucleic acidmolecule encodes a polypeptide comprising the amino acid sequence of SEQID NO:2 or SEQ ID NO:3.
 24. The nucleic acid molecule of claim 21,wherein said nucleic acid molecule is DNA.
 25. A vector comprising thenucleic acid molecule of claim
 21. 26. A cell comprising the vector ofclaim
 25. 27. A non-human transgenic animal comprising the nucleic acidmolecule of claim
 21. 28. The non-human transgenic animal of claim 27,wherein said animal is a zebrafish.
 29. A non-human animal having aknockout mutation in one or both alleles encoding a heart of glasspolypeptide.
 30. A cell from the non-human knockout animal of claim 27.31. A non-human transgenic animal comprising a nucleic acid moleculeencoding a mutant heart of glass polypeptide.
 32. The non-humantransgenic animal of claim 31, wherein the non-human transgenic animalis a zebrafish.
 33. The non-human transgenic animal of claim 35, whereinthe non-human transgenic animal comprises the heart of glass mutation.34. An antibody that specifically binds to a heart of glass polypeptide.35. Use of a compound identified using the method of claim 9 in thepreparation of a medicament for treating or preventing heart disease ina patient.
 36. Use of a heart of glass protein or an expression vectorcomprising a nucleic acid molecule encoding said protein in thepreparation of a medicament for treating or preventing heart disease ina patient.